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HEALTH  SCIENCES  STANDARD 


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Poisonous  proteins; 


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POISONOUS  PROTEINS 


POISONOUS  PKOTE1NS 


THE    HERTER   LECTURES   FOR    1916    GIVEN   IN    THE 

UNIVERSITY  AND   BELLEVUE    MEDICAL 

SCHOOL,  NEW  YORK 


BY 
VICTOR  C.  VAUGHAN,  M.D.,  LL.D. 

DEAN    OF    THE    UNIVERSITY    OP    MICHIGAN    MEDICAL    SCHOOL 


ST.  LOUIS 

THE  C.  V.  MOSBY  COMPANY 

1917 


Copyright,  1917,  By  The  C.  V.  Mosby  Company 


N^ 


Press  of 

The  C.  V.  Mosby  Company 

St.  Louis 


PREFACE 

As  stated  on  the  title  page  this  little  book  is 
made  up  of  the  Herter  Lectures  given  in  1916 
at  the  University  and  Belle vue  Medical  School, 
New  York.  These  lectures  were  published  in 
the  Journal  of  Laboratory  and  Clinical  Medi- 
cine. There  has  been  some  demand  for  re- 
prints, of  which  none  were  made.  I  have,  there- 
fore, gladly  acceded  to  the  request  of  the  pub- 
lishers, The  C.  V.  Mosby  Company,  who  have 
offered  to  present  them  in  the  present  form. 
The  researches  upon  which  they  are  founded 
have  occupied  much  of  my  time  for  many  years, 
and  have  given  me  a  concept  of  the  nature  of 
infection,  quite  different  from  that  usually 
taught. 

Hoping  that,  in  this  form,  my  studies  may  be 
helpful  to  my  fellow-workers  in  the  profession, 
I  submit  this  booklet  to  them. 

The  Authok. 

Ann  Arbor,  Mich.,  1917. 


Digitized  by  the  Internet  Archive 

in  2010  with  funding  from 
Columbia  University  Libraries 


http://www.archiye.org/details/poisonousproteinOOvaug 


CONTENTS 


PART  PAGE 

I.  Bacterial  Proteins 13 

II.  Vegetable  Proteins 45 

III.  Protein  Fever 75 

IV.  The  Purity  of  Alcohol 101 

V.  Tissue  Cellular  Protein  Poisons — The  Color 
Reactions  of  Proteins  and  Their  Split 
Products — The  Ninhydein  Reactions — The 

Protein  Skin  Reaction 105 


INTRODUCTION 

In  nature  all  proteins  are  the  products  of 
life  and  each  kind  of  living  molecule  elaborates 
and  contains  its  own  specific  protein.  Some 
forms  of  life  are  capable  of  constructing  their 
proteins  out  of  inorganic  matter,  while  others 
can  utilize  only  that  which  has  been  built  up 
by  other  cells  into  protein  material.  Plants 
take  the  ammonia,  nitrates  and  nitrites  of  the 
air,  soil  and  water,  and  by  synthetical  proc- 
esses convert  these  into  the  proteins  found  in 
their  tissues.  In  this  process  there  are  two 
stages.  In  the  first  the  inorganic  nitrogen  is 
synthesized  into  amino  acids  and  in  the  sec- 
ond these  are  combined  to  form  proteins.  The 
higher  animals  cannot  synthesize  inorganic 
nitrogen  into  amino  acids.  This  is  done  for 
them  by  plants  and  to  some  extent  by  bacteria 
in  conjunction  with  plants.  By  the  symbiotic 
action  of  certain  bacteria  and  plants  even  the 
free  nitrogen  of  the  air  is  drawn  upon  in  the 
construction  of  vegetable  proteins.  So  far  as 
protein  metabolism  is  concerned  the  vegetable 
world  is  the  synthetical  or  constructive  labora- 


10  INTRODUCTION 

tory  while  the  animal  is  the  analytical  or  de- 
structive machine.  The  plant  takes  the  small- 
est parts  and  builds  them  up  into  highly  com- 
plex bodies,  while  the  animal  takes  the  com- 
plex and  splits  them  into  pieces  to  be  recon- 
structed in  its  own  body.  In  a  general  way 
the  above  statement  is  true,  but  there  are  syn- 
thetical processes  going  on  normally  in  the 
animal  body  and  it  is  demonstrable  that  simple 
proteins  may  be  built  into  more  complex  mole- 
cules in  the  animal  body.  Moreover,  it  is  cer- 
tainly true  that  in  man  with  perfect  digestion 
practically  all  the  nitrogen  of  the  food  is 
absorbed  in  the  form  of  amino  acids.  The 
animal  as  well  as  the  plant  is  a  synthetical  lab- 
oratory, but  the  new  material  used  by  the 
-former  is  the  finished  product  of  the  latter, 
which  is  unravelled  and  then  woven  into  a  new 
pattern  which  is  different  in  each  species  of 
animal. 

There  are  as  many  kinds  of  proteins  as  there 
are  kinds  of  living  matter.  Chemically  pro- 
teins are  polymers  of  amino  acids.  The  amino 
acids  demonstrated  in  proteins  are  only  about 
eighteen  in  number,  but  with  these  put  to- 
gether in  an  almost  infinite  variety  of  ways,  we 
get  an  unlimited  variety  of  products,  just  as 
with  only  twenty- six  letters  in  the  alphabet 


INTRODUCTION  11 

there  is  no  end  to  the  making  of  words.  The 
simplest  proteins  consist  wholly  of  amino  acids. 
These  combine  with  inorganic  salts,  lime,  phos- 
phorus, iron,  etc.,  and  with  carbohydrates  to 
form  the  compound  proteins. 

All  living  things  not  only  contain  protein, 
but  this  is  their  essential  constituent.  The 
living  protein  molecule  is  in  a  labile  or  active 
state,  capable  of  trading  in  energy,  absorbing 
and  eliminating ;  never  in  a  condition  of  equilib- 
rium. Dead  protein  is  in  a  state  of  rest;  it  is 
a  stabile  molecule  and  remains  in  equilibrium. 


POISONOUS  PROTEINS 


PAET  I. 


BACTERIAL  PROTEINS. 

Material. — Fifteen  years  ago,  after  various 
attempts  to  secure  bacterial  cellular  protein  in 
large  amount,  I  succeeded  with  the  tanks  for 
massive  cultures  which  have  been  described 
elsewhere.  The  growths  thus  obtained  are 
freed  from  extraneous  matter  by  washing  with 
dilute  alcohol  and  then  by  thorough  successive 
extractions  with  absolute  alcohol  and  ether. 
The  cellular  substance  is  ground  first  in  porce- 
lain and  then  in  agate  mortars,  and  passed 
through  fine  meshed  sieves.  Whatever  the  bac- 
terium employed  the  product  is  a  fine  white 
powder.  The  dilute  alcohol  removes  the  ex- 
traneous matter  mechanically  held  by  the 
growths  and  the  long  continued  extractions  with 
alcohol  and  ether  remove  coloring  matters,  fats, 
waxes,  and  other  less  known  bodies.  I  have 
never  made  a  close  study  of  these  extractives. 
These  bacterial  powders  when  examined  micro- 
scopically show  the  individual  cells  plainly,  es- 


14  POISONOUS   PROTEINS 

pecially  when  properly  stained.  Even  the  chro- 
mogenic  bacteria  come  through  as  white  pow- 
ders, all  the  color  being  removed  by  the  alcohol 
and  ether.  The  freedom  of  this  cellular  mate- 
rial from  extraneous  matter  is  best  appreciated 
from  the  fact  that  when  one  gram  of  it  is  in- 
cinerated there  is  no  trace  of  chloride  in  the 
ash.  With  chloride  of  sodium  in  the  culture  me- 
dium and  considering  the  ease  with  which  traces 
of  chloride  are  detected,  this  indicates  a  sur- 
prising degree  of  purity  in  the  material.  The 
significance  of  the  absence  of  chloride  will  be 
discussed  later.  The  cellular  protein  of  many 
pathogenic  and  nonpathogenic  bacteria  has  been 
obtained  by  growth  in  the  tanks.  I  may  say 
that  I  have  never  dared  to  grow  anthrax  bacilli 
in  these  massive  cultures  and  have  contented 
myself  so  far  as  this  organism  is  concerned  with 
the  less  abundant  growths  in  Eoux  flasks.  For 
obtaining  abundant  growths  of  the  tubercle  ba- 
cillus the  tanks  are  less  suitable  than  glycerine 
beef-tea  cultures. 

Chemistry. — It  has  been  generally  assumed 
that  bacteria  are  unicellular  plants.  This  as- 
sumption rests,  so  far  as  I  can  learn,  upon  early 
statements,  such  as  that  of  Pollender,  that  an- 
thrax bacilli  are  not  affected  by  strong  alkali 
and  this  has  been  interpreted  as  meaning  that 


BACTEKIAL  PROTEINS  15 

they  consist  largely  of  cellulose.  It  is  true  that 
certain  investigators  have  claimed  to  demon- 
strate even  large  amounts  of  cellulose  in  bac- 
teria. Hammerschlag  on  wholly  inadequate  evi- 
dence estimated  the  per  cent  of  cellulose  in  the 
tubercle  bacillus  as  high  as  28.1.  DeSchwein- 
itz  and  Dorset  reduce  this  amount  to  6.95  per 
cent,  but  hardly  accept  this  figure  themselves 
since  they  conclude  that  cellulose  is  probably 
present  in  small  amount  in  the  tubercle  bacil- 
lus, and  not  present  in  the  bacillus  of  glanders. 
These  and  other  investigators,  who  have  re- 
ported the  presence  of  cellulose  in  bacterial  cel- 
lular substances,  have  not  properly  distin- 
guished between  cellulose  and  other  carbohy- 
drates. Vincenzi  employing  proper  tests  failed 
to  find  cellulose  in  the  cellular  substance  of 
bacillus  subtillis,  but  did  find  a  nitrogenous  car- 
bohydrate. In  our  work,  Wheeler  made  special 
search  for  cellulose  in  sarcina  lutea.  Twenty 
grams  of  cell  substance  was  autoclaved  with  25 
parts  (500  c.c.)  of  ten  per  cent  potassium  hy- 
droxide at  120°,  first  for  thirty  minutes  and 
then  for  an  hour.  There  remained  a  consider- 
able residue  which  gave  none  of  the  protein  re- 
actions, did  not  reduce  Fehling's  solution  even 
after  prolonged  boiling  with  dilute  hydro- 
chloric acid,  but  did  respond  to  the  carbohy- 


16  POISONOUS   PROTEINS 

drate  test  with  alphanaphthol.  Cellulose  could 
not  be  detected  by  any  known  test.  Schweit- 
zer's reagent  failed  to  dissolve  it  and  it  gave  no 
color  with  iodine  even  after  treatment  with  sul- 
phuric acid.  A  portion  was  dried  and  heated 
with  soda  lime  when  it  evolved  a  gas  which 
turned  red  litmus  paper  blue,  thus  indicating 
the  presence  of  nitrogen  which  had  been  re- 
duced to  ammonia.  The  odor  of  burning  feath- 
ers also  indicated  the  presence  of  nitrogen. 
From  these  results  we  conclude  that  there  is  no 
evidence  of  the  presence  of  cellulose  in  bacte- 
rial cellular  substance.  Leach  made  search  for 
cellulose  in  the  cells  of  the  colon  bacillus  with 
like  negative  results.  Like  Vincenzi  we  did  find 
a  nitrogenous  carbohydrate.  This  is  chitin  or 
some  chitin-like  substance.  The  presence  of 
chitin  in  bacterial  cell  substance  has  been  re- 
ported by  Ivanofr,  Emmerling,  Helbin,  Bulloch, 
and  others. 

My  students  and  I  have  found  two  carbohy- 
drates in  bacterial  cellular  substance.  One,  re- 
ferred to  above,  is  combined  with  nitrogen,  is 
not  soluble  in  strong  alkali,  and  does  not  reduce 
Fehling's  solution  even  after  prolonged  boiling 
with  dilute  mineral  acids.  The  second  carbo- 
hydrate is  combined  with  phosphorus,  is  soluble 
in  alkali,  and  does  reduce  Fehling's  solution 


BACTERIAL   PROTEIN'S  17 

after  being  boiled  with  dilute  mineral  acid.  In 
the  unbroken  molecule  this  carbohydrate  un- 
doubtedly is  contained  within  the  nuclein  group. 
If  the  presence  of  cellulose  be  essential  to  plant 
tissue,  bacteria  certainly  are  not  forms  of  plant 
life. 

There  is  no  controversy  concerning  the  pres- 
ence of  nuclein  in  bacterial  cellular  substance  as 
the  xanthine  bases  have  been  demonstrated 
among  the  disruption  products  both  in  my  own 
laboratory  and  elsewhere.  The  literature  on 
this  subject  is  too  extensive  to  permit  me  to 
go  into  it  exhaustively  and  I  will  content  myself 
with  a  few  references.  Klebs  obtained  from 
the  turbercle  bacillus  a  nuclein  containing  8-9 
per  cent  of  phosphorus.  From  the  same  organ- 
ism Euppel  separated  a  nuclein  containing  9.42 
per  cent  of  phosphorus  which  he  designated  tu- 
berculinic  acid.  Levin  obtained  from  the  tu- 
bercle bacillus  proteins,  nuclein,  and  crystals 
which  he  considered  a  mixture  of  thymil  and 
uracil,  also  cystosin.  Lustig  and  Galeotti  ob- 
tained a  nucleo-protein  from  the  pest  bacillus. 
In  our  work  the  presence  of  nuclein  bodies  was 
plainly  in  evidence.  Leach  obtained  from  the 
colon  cell  substance  a  body  containing  7.33  per 
cent  of  phosphorus  and  both  Leach  and  Wheeler 


18  POISONOUS   PROTEINS 

secured  evidence  of  the  presence  of  xanthine 
bases. 

Bacterial  cellular  substance  responds  to  all 
the  protein  reactions.  Proteins  are  detached 
from  the  substance  by  both  alkalis  and  acids, 
but  the  properties  of  the  bodies  thus  obtained 
indicate  that  they  are  split  products  obtained 
by  the  cleavage  of  more  complex  molecules,  and 
do  not  exist  free  in  the  cellular  substance.  Di- 
lute mineral  acid  splits  off  the  nitrogenous  car- 
bohydrate and  when  this  extraction  is  carried 
on  at  high  temperature  much  of  the  second  car- 
bohydrate is  converted  into  a  reducing  sub- 
stance. The  acid  extracts  when  dropped  into  a 
large  volume  of  alcohol  give  a  precipitate  which 
after  purification  by  resolution  in  water  and  re- 
precipitation  with  alcohol  yields  more  than 
seven  per  cent  of  phosphorus.  The  line  of 
cleavage  through  the  large  molecules  in  the  cel- 
lular substance  followed  by  acid  action  seems 
to  be  definite  and  the  same  products  are  ob- 
tained with  one  per  cent  and  0.1  per  cent  sul- 
phuric acid.  More  concentrated  acids  after 
prolonged  heating  break  deeper  into  the  molec- 
ular structure  and  cleave  the  biuret  bodies  with 
the  liberation  of  amino  acids.  "Wheeler  and 
Leach  have  made  special  studies  of  the  action 
of  mineral  acids  on  bacterial  cellular  substance. 


BACTEKIAL   PEOTEINS  19 

Ten  per  cent  solutions  of  potassium  hydroxide 
at  120°  extract  from  bacterial  cell  substance 
everything  except  the  chitin-like  body  which 
consists  of  a  carbohydrate  combined  with  nitro- 
gen. 

We  have  demonstrated  the  presence  in  bac- 
terial cellular  substance  of  both  mono  and  dia- 
mino  acids  and  have  shown  that  the  percentage 
of  these  varies  with  the  microorganism.  We 
have  found  the  percentage  of  nitrogen  to  vary 
from  5.964  in  subtilis  to  11.765  in  violaceous. 

I  began  this  work  with  the  expectation  of  find- 
ing the  bacterial  cell  substance  composed  of  rel- 
atively simple  bodies.  I  have  been  compelled  to 
come  to  the  opposite  conclusion.  The  cellular 
substance  of  bacteria  contains  highly  complex 
molecules.  We  have  demonstrated  the  presence 
of  the  following  groups  among  the  split  prod- 
ucts :  (a)  A  chitin-like  body  consisting  of  a  car- 
bohydrate combined  with  nitrogen.  It  seems 
reasonable  to  infer  that  this  exists  in  the  cellu- 
lar substance  as  a  glyco-protein.  (b)  A  car- 
bohydrate group  combined  with  phosphorus 
from  which  it  is  not  easily  detached.  This  group 
reduces  copper  after  prolonged  boiling  with  di- 
lute mineral  acid.  The  amounts  as  determined 
by  the  reduction  of  Fehling's  solution  and  cal- 
culated as  xylose  are  large,  but  we  are  not  sure 


20  POISONOUS   PROTEINS 

that  the  reducing  substance  is  all  carbohy- 
drate. Indeed,  it  might  be  better  to  speak  of 
both  of  these  groups  as  those  responding  to  the 
alphanaphthol  test  rather  than  as  carbohy- 
drates and  to  distinguish  between  them  as  non- 
reducing  and  reducing  bodies.  However,  it 
seems  clear  that  the  one  now  under  considera- 
tion is  a  subgroup  in  the  nucleinic  acid  constit- 
uent of  the  cell  substance,  (c)  The  presence  of 
nucleinic  acid  is  beyond  doubt,  as  is  shown  by 
the  high  phosphorus  content  of  some  of  the 
split  products  and  by  the  demonstration  of  the 
xanthine  bases,  (d)  That  one  or  more  protein 
groups  exist  in  the  cell  substance.  If  all  these 
groups  exist  in  the  same  molecule  the  cell  sub- 
stance must  contain  a  highly  complex  molecule 
which  would  be  best  designated  as  a  glyco- 
nucleo-protein.  The  fact  that  these  bodies  are 
removed  only  by  agents  capable  of  causing  mo- 
lecular disruption  inclines  me  to  the  belief  that 
the  molecules  which  make  the  cell  substance  are 
highly  complex.  It  may  be  said  that  this  is  an 
assumption  and  without  adequate  proof.  On 
the  other  hand,  such  a  statement  as  that  made 
by  Doerr,  that  bacterial  proteins  are  of  simple 
molecular  structure,  is  wholly  without  evi- 
dence. Because  bacteria  are  simple  morpho- 
logically is  no  proof  that  they  are  made  up  of 


BACTERIAL   PROTEINS  21 

simple  proteins.  This  certainly  is  not  true  even 
if  it  should  prove  that  I  have  overestimated  the 
size  of  these  protein  molecules. 

Tamura  working  in  Kossel's  laboratory  has 
made  a  contribution  to  the  chemistry  of  bac- 
teria. He  used  cellular  material  obtained  from 
the  bacillus  tuberculosis  and  mykobacterium 
lacticola  perrugrosum.  Both  of  these  were 
grown  in  glycerine-broth  cultures  for  five 
weeks,  then  collected  on  niters  and  washed  with 
ether  and  alcohol.  Tamura  states  that  all  the 
fats  and  waxes  cannot  be  removed  in  this  way 
and  he  resorted  to  the  following  method :  After 
partial  extraction  with  ether  and  alcohol  the 
bacterial  cells  were  rubbed  up  in  a  mortar  with 
two  parts  of  sulphuric  acid  and  one  of  water 
and  from  this  mass  extraction  with  ether  and 
alcohol  was  continued.  From  these  extracts 
Tamura  obtained  along  with  the  well  known 
fatty  acids  diamino  monophosphate,  a  sub- 
stance which  has  been  previously  found  in  egg- 
yolk,  muscle,  and  brain.  Tamura  thinks  that 
this  body  has  been  mistaken  for  lecithin  by  other 
investigators  working  with  ether  and  alcohol 
extracts  of  bacteria.  In  these  extracts  Tamura 
has  furthermore  detected  a  higher  alcohol, 
which  he  names  "mykol"  and  to  which  the 
"acid  fast"  properties  of  these  bacteria  are 


22  POISONOUS   PEOTEINS 

due.  The  statement  that  the  fats  and  waxes  in- 
cluding the  phosphatide  and  "mykol"  cannot  be 
removed  from  the  tubercle  bacillus  with  alcohol 
and  ether  without  previous  disruption  with 
strong  acid  is  erroneous.  In  our  work,  first 
published  in  1908,  we  showed  that  prolonged 
extraction  of  tubercle  cell  protein  with  alcohol 
and  ether  removed  from  the  cells  the  substance 
to  which  the  "acid-fast"  property  is  due.  We 
extracted  first  with  alcohol  in  Soxhlets  for  four 
days  and  then  with  ether  for  three  days.  There 
is  therefore  no  ground  for  the  assumption  by 
Tamura  that  either  the  phosphatide  or  the 
"  mykol "  is  in  chemical  combination  with  the 
proteins  of  the  cellular  substance.  It  is  much 
more  reasonable  to  regard  them  as  substances 
either  on  their  way  to  assimilation  into  the  cell 
molecules  or  as  excretory  products.  Tamura 
says:  "My  investigations  show  that  the  pres- 
ence of  the  diamino-phosphatide  is  not  confined 
to  the  higher  organisms.' '  This  is  another  as- 
sumption that  the  molecular  structure  of  bac- 
terial cells  is  simple  and  is  wholly  without  jus- 
tification. It  is  additional  support  of  my  con- 
tention that  chemically  the  bacterial  cell  is  high- 
ly complex  and  should  not  be  regarded  as  a 
primitive  form  of  life.  Tamura 's  work  on  the 
cellular  proteins  strongly  supports  my  claim 


BACTEEIAL   PKOTEINS  23 

that  these  are  highly  complex  in  chemical  struc- 
ture. He  states  that  he  was  not  able  to  extract 
protein  from  the  cell  substance  with  water,  salt 
solution,  or  one  per  cent  sulphuric  acid,  and 
that  even  with  alkali  a  portion  of  the  cell  sub- 
stance remained  undissolved.  Surely  this 
would  not  be  true  if  the  cell  substance  consisted 
of  simple  proteins.  He  also  obtained  the  nu- 
clein  bases,  diamino  and  mono-amino-acids.  Of 
the  last  mentioned  his  list  for  the  tubercle  ba- 
cillus contains  one  (prolin)  not  found  in  ours, 
while  ours  contains  two  (glutamic  acid  and  leu- 
cin)  not  found  in  his.  Neither  found  glycocoll 
in  the  tubercle  bacillus  while  we  found  it  in  the 
colon  bacillus. 

Tamura  concludes,  as  we  had  done  some 
years  before,  that  bacterial  cellular  substance 
contains  two  carbohydrate  groups,  but  the  one 
which  we  have  designated  as  chitin-like  body,  he 
classifies  as  a  hemicellulose.  This  name  was 
proposed  by  Schultze,  after  an  investigation  of 
various  cell  membranes,  to  include  a  group  of 
bodies,  "  which  are  wholly  soluble  on  being 
heated  with  dilute  alkalis.  In  the  cold,  five  per 
cent  sodium  hydroxide  dissolves  them  some- 
what more  slowly."  And  yet  Tamura  classes 
as  a  hemicellulose  a  body  which  remains  in  the 
residue  after  repeated  extraction  with  one  per 


24  POISONOUS   PROTEINS 

cent  sodium  hydroxide ;  besides  lie  did  not  test 
this  residue  for  nitrogen  but  seems  to  have  as- 
sumed that  this  element  was  not  present  since 
the  body  did  not  respond  to  the  protein  reac- 
tions. In  our  tests  this  body  remained  in  the 
residue  after  heating  for  one  hour  at  120°  with 
ten  per  cent  potassium  hydroxide,  and  did  not 
give  the  protein  reactions,  but  did  contain  ni- 
trogen. We  know  of  no  nitrogenous  carbohy- 
drates except  the  chitins. 

Tamura  reports  a  negative  test  for  sulphur  in 
bacterial  proteins,  but  this  is  due  to  faulty  tech- 
nic,  since  Wheeler  has  shown  that  the  sulphur 
is  masked  and  does  not  respond  to  the  ordinary 
tests,  but  its  presence  is  disclosed  when  a  por- 
tion of  the  substance  is  fused  with  metallic  so- 
dium, dissolved  in  water  and  treated  with  a 
freshly  prepared  solution  of  sodium  nitroprus- 
siate;  a  violet  color  indicating  the  presence  of 
sulphur. 

While  the  i '  acid-fast ' '  and  ' i  Gram-positive ' ' 
properties  of  certain  bacteria  depend  upon  lip- 
oids which  are  extracted  from  the  cells  by  al- 
cohol and  ether,  the  cells,  after  exhaustive  ex- 
traction with  these  solvents,  take  the  analin 
dyes  quite  as  well  or  even  better  than  before. 
For  instance,  the  extracted  tubercle  bacillus 
stains  just  as  well,  or  even  better,  than  before 


BACTERIAL  PROTEINS  25 

extraction  with  alcohol  and  ether,  bnt  now  the 
stain  is  easily  removed  by  dilute  acid.  This  be- 
havior of  bacterial  cellular  substance  towards 
basic  analin  dyes  quite  naturally  suggests  that 
the  former  consists  largely  of  nuclear  material. 
In  my  opinion  this  is  strengthened  by  the  stud- 
ies of  the  cellular  substance  which  I  have  out- 
lined. Additional  evidence  in  the  same  direc- 
tion is  not  wanting.  When  sporogenous  bac- 
teria form  spores  or  pass  into  the  resting  stage 
the  essential  part  of  the  bacterial  cell  is  con- 
tained in  the  spores  and  all  spores  and  repro- 
ductive cells  consist  in  part  at  least  of  nuclear 
material.  Certain  bacteria  which  do  not  form 
spores  pass  into  a  granular  state  in  which  po- 
tential life  continues  for  a  long  time.  For  in- 
stance, the  bacillus  of  glanders,  though  an  aspo- 
rogenous  organism,  may  retain  viability  for  a 
long  time.  Wladmiroff  states  that  he  found 
these  organisms  in  glycerine-bouillon  tubes, 
with  the  ordinary  cotton  plug  capable  of  growth 
after  standing  four  years.  The  same  is  true  of 
the  plague  bacillus.  This  phenomenon  is  ex- 
plainable only  on  the  assumption  that  these  ba- 
cilli contain  nuclein.  However,  assumption  is 
no  longer  necessary  since  nuclein,  nucleinic  acid 
and  their  derivatives  have  been  found  in  all 
bacterial  cells  submitted  to  chemical  study.    I 


26  POISONOUS   PROTEINS 

am  strongly  of  the  opinion  that  the  bacterial 
cellular  substance  as  I  have  prepared  and  stud- 
ied it,  freed  from  the  extractives  soluble  in  wa- 
ter, salt  solution,  alcohol  and  ether,  is  practi- 
cally all  nuclear  material. 

Some  years  ago  A.  B.  Macallum  by  micro- 
chemical  methods  showed  that  nuclei  are  free 
from  chlorine.  His  statement  is  as  follows: 
"Intercellular  material  and  structures,  includ- 
ing the  so-called  cement  substance  of  von  Eech- 
linghausen  are  rich  in  chlorides  but  normal  nu- 
clei of  animal  and  vegetable  cells  are  absolutely 
free  from  them."  When  my  coworkers  found 
no  chlorine  in  the  ash  of  our  cellular  substance, 
I  thought  that  this  must  be  due  to  careless  work. 
I  could  not  believe  that  with  the  chlorides,  espe- 
cially sodium  chloride,  as  abundantly  distrib- 
uted as  they  are,  they  could  be  wholly  want- 
ing in  this  material,  but  repeated  examinations 
confirmed  the  first  finding.  The  finding  of  chlo- 
rine would  not  have  been  a  conclusive  evidence 
that  the  material  is  not  wholly  nuclear,  but  the 
failure  to  find  any  trace  of  this  element  I  re- 
gard as  most  convincing  evidence  that  it  is 
wholly  nuclear.  Furthermore,  Macallum  found 
that  the  phosphorus  and  iron  in  nuclear  mate- 
rial are  masked,  that  is,  they  camiot  be  detected 
without  more  or  less  marked  disruption  of  the 


BACTERIAL   PROTEINS  27 

molecule.  This  is  true  Qf  our  cellular  substance. 
I  may  recall  the  further  fact  that  the  sulphur 
is  so  masked  that  even  in  the  laboratory  of  so 
eminent  a  chemist  as  Kossel  it  was  not  detected 
in  bacterial  cellular  substance.  It  is  well  known 
that  in  proteins  sulphur  exists  in  two  forms,  one 
being  readily  split  off  with  dilute  alkali  forming 
a  sulphide,  while  the  other  is  obtained  only 
when  the  disruption  of  the  protein  molecule  is 
carried  much  further.  The  former  is  wanting 
and  the  latter  present  in  our  cellular  substance. 
I  do  not  suppose  that  all  nuclear  material  has 
the  same  elemental  constituents,  indeed,  it  is 
not  supposable  that  this  is  true,  but  the  above 
facts  seem  worthy  of  consideration. 

The  laborious  and  valuable  researches  of 
Macallum  have  shown  that  nonnucleated  or- 
ganisms, such  as  cyanophycea,  beggiota  and 
yeast  cells,  contain  nuclein,  and  this  is  prob- 
ably true  of  every  cell  which  is  capable  of  re- 
production. We  are  no  longer  quite  willing  to 
accept  the  dictum  of  Schultze,  Hertwig  and 
others  that  every  cell  must  contain  a  morpho- 
logically recognizable  part,  known  as  a  nu- 
cleus. We  may  insist  upon  the  presence  of  nu- 
clear matter,  but  not  of  nuclei.  Some  morpholo- 
ogists  have  seen  the  necessity  of  altering  our 
conception  of  a  cell.    Bourne  has  proposed  that 


28  POISONOUS  PROTEINS 

Sclmltze's  definition  be  changed  to  read:  "A 
cell  is  a  corpuscle  of  protoplasm  which  contains 
a  specialized  element,  nuclein." 

It  should  be  understood  that  the  cellular  sub- 
stance which  I  have  been  discussing  is  not  iden- 
tical with  that  which  exists  in  the  living,  mul- 
tiplying bacteria.  The  latter  consists  of  the 
former  with  the  addition  of  all  the  extractives 
which  I  have  removed  by  the  solvents,  such  as 
water,  dilute  alcohol,  absolute  alcohol  and  ether. 
The  living  bacillus  has  been  stripped  of  all  its 
surrounding  food  supplies,  its  accumulated  ex- 
cretory products  and  its  storehouse  of  fats, 
waxes,  etc.  I  have  a  strong  suspicion  that  in 
some  of  our  bacterial  reactions,  notably  with 
precipitins  and  agglutinins,  these  extractives 
are  concerned,  while  the  cellular  constituents 
have  no  direct  part.  The  active  constituents  of 
the  culture,  the  agglutinable  substance,  is  not, 
in  my  opinion,  an  essential  constituent  of  the 
bacterial  cells,  but  consists  of  one  or  more  pro- 
teins closely  associated  with  the  bacterial  cells. 
It  may  be  a  protein  already  split  off  from  the 
surrounding  pabulum  preparatory  to  absorp- 
tion and  assimilation,  or  it  may  be  an  excretory 
product.  My  reasons  may  be  stated  as  follows : 
(1)  Agglutination  does  not  destroy  the  viabil- 
ity or  virulence  of  bacteria;  therefore,  the  re- 


BACTEKIAL   PROTEINS  29 

action  does  not  disrupt  the  living  bacterial  cell. 
(2)  Thoroughly  washed  typhoid  bacilli  are  not 
agglutinable.  (3)  When  typhoid  bacilli  are 
thoroughly  shaken  in  salt  solution  so  as  to  re- 
move their  flagellar  and  the  bacilli  are  deposited 
in  a  centrifuge,  the  emulsion  of  flagellar  is  ag- 
glutinable. (4)  Neufeld  has  shown  that  when 
cholera  bacilli  are  thoroughly  cleansed  by  be- 
ing shaken  with  one  per  cent  alkali,  which  does 
not  destroy  them  and  only  washes  away  adher- 
ent matter,  they  are  not  inagglutinable  but  pro- 
duce no  agglutinin  when  injected  into  animals. 

Agglutination  and  precipitation  are  closely 
related  phenomena.  When  a  bacterial  culture 
is  filtered,  some  of  the  proteins  about  the  cells 
pass  into  solution  and  constitute  the  precipito- 
gen  while  some  of  the  same  class  of  near-cell 
proteins  remain  adherent  to  the  cells  and  consti- 
tute the  agglutinable  substance  or  the  agglutin- 
ogen. 

I  find  that  the  bacterial  cellular  substances  on 
standing  undergo  autolytic  cleavage.  We  are 
just  now  examining  a  bottle  of  colon  cellular 
substance  which  was  prepared  ten  years  ago. 
It  was  only  air  dried  and  contains  a  small 
amount  of  moisture.  When  freshly  prepared 
water  or  salt  solution  extracted  no  protein,  now 


30  POISONOUS   PKOTEINS 

one-third  the  nitrogen  passes  into  solution  when 
the  substance  is  treated  with  these  solvents. 

Poisonous  Action. — We  have  found  all  the 
bacterial  cellular  proteins  poisonous.  Our  ear- 
lier work  was  done  with  the  colon  cell  substance. 
Since  all  of  these  bodies  are  insoluble  in  water 
or  salt- solution,  it  has  been  necessary  to  admin- 
ister them  in  suspension.  Early  studies  demon- 
strated the  following  facts:  (1)  The  poison  is 
contained  within  the  bacterial  cell  and  does  not 
under  ordinary  conditions  diffuse  into  the  cul- 
ture medium.*  It  is  true  that  old  cultures  may 
contain  soluble  poisons,  but  these  result  from 
autolysis.  (2)  The  poison  is  not  extracted  from 
the  cellular  substance  by  water,  saline  solution, 
alcohol  or  ether,  either  at  ordinary  temperature 
or  at  the  boiling  point.  (3)  Heating,  even  to 
140°  in  the  autoclave  does  not  destroy  the 
poison.  (4)  Dilute  (0.5  per  cent)  solutions  of 
caustic  alkali  disrupt  the  cellular  substance 
slowly  and  imperfectly.  Stronger  (2  per  cent) 
solutions  break  up  the  cell  substance  and  render 
the  poisonous  fraction  soluble.  (5)  Boiling 
with  dilute  mineral  acid  (to  1  per  cent)  has  but 
little  effect. 

At  first  we  were  much  puzzled  by  the  fact  that 
smaller  doses  killed  while  larger  ones  failed  to 

*It  is  understood  that  we  are  speaking  of  cellular  poisons  and  not  of 
bacterial  toxins. 


BACTEEIAL   PEOTEIXS  31 

do  so.  This  was  observed  when  we  were  ad- 
ministering the  substance  by  intraperitoneal  in- 
jection. Then  we  found  that  the  more  finely 
the  substance  was  ground  the  smaller  was  the 
fatal  dose.  When  the  snbstance  was  only 
coarsely  ground  in  a  porcelain  mortar  and  sus- 
pended in  water  it  did  not  kill  guinea-pigs  on  in- 
traperitoneal injection  in  doses  less  than  1  to 
40,000  parts  body  weight.  When  the  same  pow- 
der was  more  finely  ground  in  an  agate  mortar, 
it  killed  15  out  of  16  animals  at  1  to  75,000 ;  9 
out  of  28  at  1  to  100,000;  5  out  of  8  at  1  to 
200,000;  4  out  of  34  at  1  to  2,000,000  body 
weight.  We  observed  that  when  heavy  suspen- 
sions were  used,  lumps  of  the  substance  re- 
mained undissolved  in  the  peritoneal  cavity  af- 
ter death  or  recovery.  In  these  observations 
we  found  the  solution  of  our  puzzle.  The  poi- 
sonous action  of  the  cellular  substance  is  in  pro- 
portion to  the  extent  to  which  and  the  rapidity 
with  which,  it  is  split  up  by  the  secretions  of  the 
body  cells  and  this  cleavage  is  determined  by 
the  relative  surface  exposure  of  the  substance 
to  the  action  of  the  cleavage  agents.  I  dare 
say  that  the  difference  in  susceptibility  as 
shown  among  the  individual  animals  is  due  to 
the  abundance  and  effectiveness  of  the  secre- 
tions elaborated  by  the  body  cells. 


32  POISONOUS   PROTEINS 

As  lias  been  said,  we  found  the  cellular  pro- 
teins of  all  the  bacteria  studied  more  or  less 
harmful  to  animals  when  introduced  parente- 
rally.  The  size  of  the  dose  necessary  to  pro- 
duce a  fatal  result  varies  greatly  with  the 
source  of  the  protein.  The  cellular  substance  of 
bacteria  to  which  in  its  living  state  an  animal 
is  highly  susceptible  does  not  kill  that  animal 
at  all  or  does  so  only  after  large  doses.  We 
have  injected  into  the  abdominal  cavities  of 
guinea-pigs  the  cellular  proteins  of  the  tuber- 
cle bacillus  in  quantities  of  from  five  to  two 
hundred  mg.  without  causing  death  in  a  single 
instance,  while  on  the  other  hand  a  fraction  of 
a  mg.  of  the  protein  from  bacillus  prodigiosus 
kills.  To  kill  a  guinea-pig  one  part  of  the  cel- 
lular substance  of  the  anthrax  bacillus  to  1,700 
parts  of  body  weight  is  necessary,  while  with 
the  colon  substance  one  part  to  75,000  kills  all 
animals  provided  the  material  is  finely  ground. 
In  general  it  may  be  said  that  the  more  highly 
susceptible  a  given  animal  is  to  infection  with 
a  given  bacterium  the  more  difficult  it  is  to  kill 
that  animal  with  the  cellular  protein  of  that 
bacterium.  On  the  other  hand,  the  more  highly 
immune  a  given  animal  to  infection  with  a  given 
bacterium  the  more  readily  does  that  animal 
succumb  to  injections  of  the  cellular  proteins 


BACTERIAL   PROTEINS  33 

of  that  bacterium.  At  first  sight  these  state- 
ments seem  wholly  irrational,  but  when  we 
study  them  we  find  that  they  are  not  only  rea- 
sonable but  in  accord  with  what  might  have 
been  reasonably  predicted  beforehand.  The 
guinea-pig  is  highly  susceptible  to  infection 
with  the  tubercle  bacillus  because  the  secretions 
of  its  body  cells  have  no  destructive  action  on 
that  organism.  This  together  with  the  fact  that 
the  bacillus  tuberculosis  can  feed  upon  certain 
proteins  in  the  guinea-pig's  body  are  the  essen- 
tial factors  in  the  susceptibility.  The  infecting 
bacillus  finds  an  abundance  of  suitable  food  and 
meets  with  no  resistance.  On  the  other  hand 
the  guinea-pig  is  highly  immune  to  infection 
with  the  bacillus  prodigiosus  because  the  ani- 
mal's body  cells  supply  secretions  which  are 
immediately  destructive  to  this  organism  and 
the  first  of  these  bacilli  finding  their  way  into 
the  animal's  body  meet  with  immediate  and 
complete  annihilation.  But  when  the  prodigio- 
sus is  grown  in  vitro  and  a  sufficient  amount  of 
its  cellular  substance,  dead  or  alive,  is  thrown 
into  the  abdominal  cavity  the  same  agency 
which  has  given  the  animal  immunity  to  infec- 
tion now  causes  it  to  fall  a  victim  to  the  protein 
poison.  These  facts  are  of  practical  as  well  as 
scientific    interest    because    they   undoubtedly 


34  POISONOUS   PROTEINS 

form  the  basis  of  the  frequently  reported  and 
well  attested  observations  of  some  of  the  great 
clinicians  of  the  past  that  the  case  mortality  in 
certain  infections,  most  notably  in  typhus  fever, 
is  much  higher  in  the  better  nourished  than  in 
the  less  robust. 

As  I  have  indicated  the  cellular  proteins  when 
introduced  parenterally  into  animals  are  not 
wholly  harmless  even  when  they  do  not  kill. 
When  the  cellular  substance  of  the  bacillus  tu- 
berculosis is  injected  into  the  abdominal  cav- 
ity of  a  guinea-pig  it  has  no  recognizable  ef- 
fect so  far  as  the  behavior  or  external  condition 
of  the  animal  shows.  The  dead  bacilli  are  taken 
up  in  the  folds  of  omentum  and  develop  local 
tubercles.  When  the  cellular  substance  of  the 
colon  bacillus  is  injected,  a  peritonitis  results. 
In  short,  the  lesions  which  follow  infections  re- 
sult also  from  the  injection  of  the  dead  cellular 
substance.  I  conclude  from  this  that  the  lesions 
of  the  infections  are  not  due  to  the  activity  of 
the  living  bacilli,  but  result  from  reaction  be- 
tween the  bacterial  proteins  and  the  body  cells. 

Split  Peoducts. — In  1903,  Wheeler  and  1 
found  that  the  bacterial  cellular  proteins  could 
be  split  into  poisonous  and  nonpoisonous  parts 
and  later  we  showed  that  all  true  proteins  can 
be  broken  up  in  the  same  way.    This  work  has 


BACTERIAL  PROTEIN'S  35 

been  confirmed  by  many  investigators.  There 
are  several  ways  in  which  this  cleavage  can  be 
secured,  bnt  the  most  satisfactory  is  the  one 
which  we  first  employed.  The  dried  protein, 
after  exhaustive  extraction  with  alcohol  and 
ether,  is  repeatedly  heated  at  78°  with  a  two  per 
cent  solution  of  sodium  hydroxide  in  absolute 
alcohol.  When  this  is  done  the  poisonous  frac- 
tion goes  into  solution  while  the  nonpoisonous 
part  remains  undissolved  and  is  removed  by 
filtration.  This  is  evidently  a  true  cleavage  and 
not  a  mere  disintegration.  The  nonpoisonous 
portion  contains  all  the  carbohydrate  and  phos- 
phorus of  the  original  complex  molecule. 

The  Protein  Poison. — Since  this  body  has 
been  obtained  from  all  true  proteins,  bacterial, 
vegetable  and  animal,  so  far  examined,  we  have 
called  it  "the  crude  soluble  poison;"  "crude" 
because  it  is  undoubtedly  a  mixture  of  chemical 
bodies  and  "soluble"  in  contradistinction  to  the 
bacterial  cellular  proteins  from  which  it  was 
first  prepared.  Aqueous  solutions  are  somewhat 
opalescent,  and  may  be  quite  turbid.  Filtration 
through  hard  paper  generally  gives  a  clear  fil- 
trate but  with  some  preparations  we  have  found 
filtration  through  porcelain  necessary  to  secure 
a  perfectly  clear  solution.  All  the  crude  soluble 
poisons  that  we  have  obtained  give  the  biuret 


36  POISONOUS   PROTEINS 

and  Millon  tests.  None  give  the  Molisch  test, 
thns  showing  the  absence  of  carbohydrate. 
Some  give  the  Adamkiewicz  and  Liebermann 
tests  while  others  do  not.  This  test  is  believed 
to  be  due  to  the  presence  of  tryptophane.  The 
fact  that  the  poisons  from  certain  proteins  do 
not  respond  to  these  tests  indicates  that 
Doerr's  assumption  that  the  poisonous  action  is 
due  to  the  presence  of  this  group  is  without 
support.  The  poison  gives  the  Millon  test  most 
strikingly  and  in  high  dilution.  This  test  is  be- 
lieved to  indicate  the  presence  of  tyrosine  and 
it  is  interesting  to  note  that  gelatine  which  con- 
tains no  tyrosine  does  not  yield  the  poison. 
Aqueous  solutions  are  distinctly  acid  to  litmus 
and  this  reaction  is  due  to  some  organic  body. 
Neutralization  with  alkalis  and  alkaline  earths 
weaken  the  action  of  the  poisons.  Poisons  from 
some  proteins  appear  to  form  definite  com- 
pounds with  calcium  and  magnesium  and  at 
least  some  of  the  calcium  bodies  are  inert.  In 
the  dry  state  the  protein  poison  forms  a  brown- 
ish powder  varying  somewhat  in  shade  with  the 
protein  from  which  it  is  obtained.  All  prepara- 
tions have  the  same  marked  odor.  It  is  much 
mjore  freely  soluble  in  absolute  alcohol  than  in 
water.  Whether  it  should  be  called  a  protein  or 
not  is  a  question.    Proteins  should  not  be  solu- 


BACTERIAL   PROTEINS  37 

ble  in  absolute  alcohol.  However  this  substance 
gives  the  biuret  test  and  this  is  generally  re- 
garded as  the  most  distinctive  test  for  proteins. 
Its  alcoholic  solutions  are  precipitated  by  alco- 
holic solutions  of  copper,  mercury  and  plati- 
num. By  means  of  these  precipitants  with  sub- 
sequent removal  of  the  metal  with  hydrogen 
sulphide,  we  have  obtained  our  most  potent 
preparations.  By  this  method  we  have  ob- 
tained a  body  which  kills  guinea-pigs  of  from 
two  hundred  to  three  hundred  grams  weight  in 
doses  of  0.5  mg.  given  intravenously.  The 
poison  is  not  an  alkaloid,  although  it  may  be 
basic  in  character. 

Action  on  Animals. — The  comparative  ef- 
fects of  the  living  bacillus,  the  dead  cellular 
substance  and  the  crude  soluble  poison  on  ani- 
mals was  first  worked  out  by  V.  C.  Vaughan, 
Jr.    The  organism  used  was  the  colon  bacillus. 

(a)  The  Living  Bacillus. — When  a  guinea- 
pig  receives  a  fatal  dose  of  the  living  colon 
bacillus  intraperitoneally  there  is  a  period  of 
from  five  to  twelve  hours,  varying  with  the  size 
of  the  inoculation,  during  which  there  are  no 
recognizable  symptoms.  We  regard  this  as  the 
period  of  incubation,  and  it  is  roughly  propor- 
tional to  the  amount  of  the  culture  used  and  to 
some  extent  to  the  virulence  of  the  organism  or 


38  POISONOUS   PROTEINS 

the  rate  at  which  the  bacillus  multiples.  This 
work  was  done  with  a  bacillus,  1  c.c.  of  a  twelve 
hour  or  older  bouillon  culture  of  which  invari- 
ably killed  within  twenty-four  hours.  When 
this  amount  was  given  no  effects  became  visible 
for  a  period  of  from  ten  to  twelve  hours.  With 
larger  doses  the  period  of  incubation  was  some- 
what shorter,  but  with  the  largest  doses  of  the 
richest  cultures  there  is  still  a  period  of  incu- 
bation. This  measures  the  time  necessary  for 
two  things  to  happen.  First  the  bacillus  must 
multiply  sufficiently  to  supply  enough  poison  to 
visibly  affect  the  animal.  Second,  this  poison 
must  be  made  effective  by  being  split  out  of  the 
large  molecule  of  which  it  is  a  part.  Therefore, 
while  the  period  of  incubation  is  not  accompa- 
nied by  the  development  of  symptoms  which 
rise  to  the  plane  of  observation,  it  is  actually  a 
critical  period  in  every  infection  and  the  out- 
come depends  upon  whether  the  bacteria  are  all 
destroyed  before  a  lethal  dose  of  the  poison  has 
been  developed  by  the  multiplication  of  the  ba- 
cillus and  set  free  or  made  effective  by  the  se- 
cretions of  the  body  cells.  It  is  during  this 
period  that  natural  and  acquired  immunity 
either  save  the  day  or,  for  the  time  at  least,  fail. 
In  natural  infection  the  number  of  bacilli  in- 
troduced is  small  and  in  case  of  full  immunity 


BACTERIAL   PROTEINS  39 

these  are  all  destroyed,  there  is  no  multiplica- 
tion and  the  amount  of  poison  set  free  in  the 
destruction  of  the  small  number  of  the  invaders 
is  not  sufficient  to  induce  symptoms  or  to  de- 
velop lesions.  This  is  what  happens  when  the 
smallpox  virus  finds  its  way  into  the  body  of 
one  thoroughly  immunized  by  a  previous  attack 
of  the  disease  or  by  successful  vaccination. 
When  the  immiinity  is  only  partial  or  when  the 
infection  is  massive  or  unusually  virulent, ,  the 
virus  develops  for  a  time,  becomes  more  or  less 
distributed  in  certain  tissues  and  its  final  de- 
struction is  accompanied  by  the  development  of 
symptoms,  and  the  reaction  between  the  virus 
and  the  body  cells  leaves  more  or  less  marked 
lesions.  When  there  is  no  immunity  the  virus 
multiplies  without  hindrance  and  life  is  de- 
stroyed. There  are  infections  in  which  the 
body  shows  little  or  no  resistance.  Some  of 
these  run  an  acute  course  and  destroy  life  in  a 
few  days,  while  others  are  more  chronic.  This 
seems  to  depend  upon  the  rate  of  multiplication 
in  the  invading  organism.  Apparently  there  is 
relatively  as  much  difference  in  the  rate  of  mul- 
tiplication in  bacteria  as  there  is  among  the 
higher  animals.  The  "generation  period"  or 
the  interval  between  fissions  varies  among  spe- 
cies and  strains,  and  is  influenced  by  external 


40  POISONOUS   PEOTEINS 

conditions.  Virulence  is  largely  determined  by 
rate  of  multiplication  or  at  least  the  two  cor- 
respond. Under  favorable  conditions  the  chol- 
era bacillus  divides  abont  every  half  hour.  So 
far  as  I  know  no  one  has  determined  the  "gen- 
eration period"  in  the  tubercle  bacillus,  but  it 
is  certainly  much  longer.  It  follows  that  cholera 
is  an  acute  disease,  often  terminating  fatally  in 
a  few  hours  while  tuberculosis  extends  through 
months  and  even  years.  The  guinea-pig  shows 
no  resistance  to  the  tubercle  bacillus  and  the  or- 
ganism slowly  but  steadily  grows,  develops  its 
characteristic  lesions  and  kills,  probably 
through  its  autolytic  products  and  without  de- 
veloping any  antagonistic  action  in  the  body 
cells.  Eodents,  especially  rats,  show  but  little 
or  no  resistance  to  the  plague  bacillus,  except  in 
those  regions  where  this  disease  is  endemic  and 
there,  it  is  said,  this  disease  even  among  the 
rats  becomes  a  chronic  infection. 

Our  intraperitoneal  infection  of  the  guinea- 
pig  is  comparable  with  the  development  of  a 
general  peritonitis  from  a  ruptured  appendix. 
The  period  of  incubation  is  short  and  while 
there  may  be  some  elevation  of  temperature, 
this  is  not  marked  or  even  constant.  During 
the  period  of  incubation,  when  the  bacilli  are 
abundantly    multiplying,  the  behavior  of  the 


BACTERIAL   PROTEINS  41 

animal  in  no  way  distinguishes  it  from  its  un- 
treated fellows,  but  at  the  end  of  this  period 
there  is  a  marked  change.  The  animal  no 
longer  eats;  its  coat  becomes  rough;  its  head 
droops;  it  sits  in  one  corner  of  the  cage  in  a 
stupor;  its  abdominal  walls  become  rigid  and 
pressure  over  this  region  elicits  evidence  of 
pain.  Now,  its  temperature  begins  to  fall  and 
this  decline  is  progressive  in  fatal  cases.  We 
have  frequently  seen  the  temperature  fall  from 
101°  to  94°  in  from  two  to  four  hours  and  it 
may  reach  85°  and  even  lower  before  death.  A 
rise  in  temperature  after  it  begins  to  fall  gen- 
erally means  recovery.  Autopsy  reveals  a  gen- 
eral hemorrhagic  peritonitis  with  a  large 
amount  of  bloody  fluid  with  intact  red  corpus- 
cles and  leucocytes  in  the  peritoneal  cavity. 
The  parietal  and  visceral  peritoneum  are  stud- 
ded with  minute  punctiform  hemorrhages  and 
there  is  more  abundant  hemorrhage  in  the  great 
omentum.  The  chemotactic  pull  of  the  bacilli 
has  been  not  only  great  enough  to  assemble 
great  numbers  of  leucocytes,  but  violent  enough 
to  rupture  small  blood  vessels. 

(b)  The  Cellular  Proteins. — When  a  fatal 
quantity  of  the  cellular  protein  of  the  colon  ba- 
cillus is  injected  into  the  peritoneal  cavity  of 
a  guinea-pig  the  progress  of  events  is  exactly 


42  POISONOUS   PKOTEINS 

like  that  following  infection  with  the  living  or- 
ganism except  that  the  period  of  incubation  is 
shortened.  There  is  no  longer  either  opportu- 
nity or  need  for  the  multiplication  of  the  bacil- 
lus. This  has  taken  place  in  vitro  and  enough 
of  the  protein  to  kill  has  been  introduced.  One 
of  the  features  that  characterizes  and  marks  the 
period  of  incubation  has  been  withdrawn.  It 
only  remains  for  the  body  cells  by  means  of 
their  secretions  to  cleave  the  bacterial  protein 
and  set  the  poison  free.  The  period  of  incu- 
bation is  reduced  half  or  more,  then  the  evi- 
dences of  poisonous  action  are  exactly  the  same 
as  in  the  inoculated  animal.  The  temperature 
falls  at  the  same  rate  and  autopsy  reveals  ex- 
actly the  same  lesions.  The  chemotactic  pull  of 
the  dead  protein  has  proved  just  as  strong  and 
just  as  violent  as  that  of  the  living  protein.  In 
fact  the  pull  in  both  instances  is  a  chemical  and 
not  a  vital  one  and  the  lesions  result  from  a  re- 
action between  the  proteins  of  the  bacterial  cells 
and  those  of  the  body  cells. 

(c)  The  Soluble  Poison. — When  a  fatal  dose 
of  the  crude  soluble  poison  is  injected  into 
the  peritoneal  cavity,  the  effects  begin  to  reveal 
themselves  much  sooner.  There  is  now  no  pe- 
riod of  incubation.  Both  steps,  which  have 
characterized  this  period,  are  now  omitted.  The 


BACTERIAL   PROTEINS  43 

bacillus  has  been  grown  and  has  been  cleaved  in 
vitro.  The  action  of  the  poison  begins  to  mani- 
fest itself  within  a  few  minutes — from  five  to 
twenty — and  it  appears  in  three  well  marked 
stages :  The  first  we  have  designated  as  that  of 
peripheral  irritation.  In  the  guinea-pig,  it  is 
manifest  by  the  animal  scratching  itself,  gen- 
erally first  on  the  nose  and  then  over  every  part 
of  the  body  which  can  be  reached  by  its  claws. 
In  man,  an  erythematous  blush,  beginning  about 
the  point  of  injection,  spreads  over  the  body 
and  may  be  followed  by  an  urticarial  rash  with 
intense  itching.  This  is  not  always  confined  to 
the  cutaneous  surface,  but  may  extend  to  the 
mucous  membrane  of  the  mouth,  throat  and  rec- 
tum. The  second  stage  is  one  of  partial  paraly- 
sis. The  guinea-pig  lies  on  its  side,  with  rapid, 
shallow  and  difficult  breathing.  When  urged  to 
move,  it  shows  inability  to  coordinate  its  move- 
ments and  partial  paralysis  is  evident,  especial- 
ly in  the  posterior  extremities.  In  man,  the 
breathing  becomes  distressingly  asthmatic. 
Air-hunger  is  marked  and  there  is  a  sense  of 
impending  danger.  The  convulsive  stage  marks 
the  termination.  The  convulsions  are  usually 
clonic  and  at  first  generally  involve  only  the 
neck  muscles,  the  head  being  thrown  back.  The 
seizures  extend  over  the  body,  becoming  more 


44  POISONOUS   PEOTEINS 

frequent  and  violent.  During  a  convulsion,  oc- 
casionally in  an  interval,  respiration  ceases. 
The  heart  continues  to  beat,  at  first  with  no  ac- 
celeration and  with  perfect  regularity.  The  ex- 
act mode  differs  somewhat  in  different  animals, 
but  is  always  that  of  anaphylactic  shock.  Ne- 
cropsy shows  the  same  conditions  found  after 
death  from  anaphylactic  shock.  The  peritonitis 
found  after  death  from  inoculation  or  from  the 
injection  of  the  unbroken  cellular  protein  is 
wholly  wanting. 

"When  a  nonfatal  dose  of  the  soluble  poison  is 
administered,  the  symptoms  are  those  described 
above  as  characterizing  the  first  and  second 
stages.  There  may  be  isolated  and  slight  con- 
vulsive seizures,  but  an  animal  seldom  recovers 
after  the  convulsions  have  become  general  and 
frequent.  With  recovery  the  temperature  slow- 
ly rises  and  ultimately  returns  to  normal. 
Within  two  hours  the  animal  is  apparently  quite 
normal  in  every  respect. 


PAET  II. 

VEGETABLE  PEOTEINS. 

Thanks  to  the  researches  of  Osborne,  a  nuim 
ber  of  vegetable  proteins  may  be  obtained  in  a 
pure  state  and  in  quantity.  The  work  done  in 
my  laboratory  was  npon  some  of  the  seed  pro- 
teins, especially  zein  from  cornmeal  and  ede- 
stin  from  hemp  seed,  which  were  prepared  by 
Leach  according  to  the  methods  of  Osborne. 
From  these  proteins  we  split  off  the  protein 
poison  by  the  same  process  employed  in  the 
cleavage  of  the  bacterial  proteins.  The  poisons 
obtained  from  zein  and  edestin  showed  no  dif- 
ference either  in  response  to  chemical  tests  or 
in  physiological  action  from  those  obtained 
from  the  cellular  substance  of  bacteria.  My 
present  purpose  in  bringing  out  these  facts  lies 
in  the  evidence  which  they  bear  in  support  of 
my  contention  that  the  protein  poison  is  a 
group  in  the  protein  molecule  and  that  it  is 
present  in  all  true  proteins.  So  long  as  my 
studies  were  confined  to  the  highly  complex  bac- 
terial proteins,  I  was  not  sure  of  the  correctness 
of  this  idea.    With  edestin  we  are  supposed  to 


46  POISONOUS  PKOTEINS 

have  an  unmixed  protein.  It  is  a  single  com- 
pound, of  highly  complex  structure  it  is  true, 
but  not  a  mixture  of  different  molecules.  If  the 
poison  be  detached  from  this  by  chemical  cleav- 
age it  must  follow  that  the  poison  consists  of 
a  group  which  exists  within  the  larger  body. 
The  importance  of  this  will  be  more  evident 
when  I  call  attention  to  the  fact  that  some  years 
ago  Pick  and  Spiro  were  unable  to  obtain  from 
edestin  the  substance  which  when  injected  into 
animals  retards  the  coagulation  of  the  blood 
and  from  this  failure  they  concluded  that  this 
body  is  not  a  true  cleavage  product  of  proteins 
and  that  it  is  not  an  intramolecular  constituent 
of  pure  proteins.  In  fact  they  came  to  the  con- 
clusion that  the  coagulation-retarding  substance 
is  neither  a  protein  nor  a  protein  derivative. 
The  relation  between  the  protein  poison  and  the 
coagulation-retarding  substance  will  be  dis- 
cussed later.  At  this  point  I  simply  wish  to 
emphasize  my  claim  that  the  protein  poison  is 
an  intramolecular  constituent  of  proteins  and 
that  it  is  obtained  by  the  chemical  cleavage  of 
protein  molecules.  Edestin  being  a  simpler 
and  smaller  molecule  than  bacterial  cellular 
substance  is  the  more  suitable  matrix  from 
which  the  protein  poison  may  be  obtained.  The 
yield  is  larger  and  the  by-products  less  in  va- 


ANIMAL   PEOTEINS  47 

riety  and  abundance.  Edestin  contains  no  car- 
bohydrate group  while  bacterial  proteins  con- 
tain two  and  these  gave  ns  much  trouble  in  onr 
earlier  attempts  to  isolate  the  poison. 

Animal  Proteins. 

We  have  prepared  the  protein  poison  from 
a  great  number  and  variety  of  animal  proteins, 
such  as  egg-white,  casein,  serum  albumin,  se- 
rum globulin,  blood  cells,  muscle,  brain,  liver, 
kidney,  etc.  In  fact,  we  have  found  no  true  pro- 
tein which  does  not  yield  the  poison  when  split 
up  by  the  method  given — a  two  per  cent  solu- 
tion of  sodium  or  potassium  hydroxide  in  ab- 
solute alcohol. 

In  beginning  this  work  I  expected  to  find  the 
simplest  proteins  in  unicellular  organisms.  As 
I  have  already  indicated  this  expectation  has 
not  been  realized.  The  proteins  of  most  simple 
structure  I  have  found  in  seeds  and  in  the 
casein  of  milk.  Seeds  contain  the  embryo  ac- 
companied by  simple  proteins  and  varying 
amounts  of  fat  and  carbohydrate,  also  proteo- 
lytic, amylolytic  and  lipolytic  ferments.  When 
the  seeds  are  placed  under  proper  conditions  of 
temperature  and  moisture,  the  ferments  begin 
to  act,  the  storehouses  of  foods  are  split  into 
available  building  blocks  and  growth  begins.  In 


48  POISONOUS   PKOTEINS 

milk  the  food  supply  for  the  young  is  supplied 
in  similar  form.  The  carbohydrate  exists  in 
the  form  of  milk  sugar.  The  fat  exists  as  such. 
The  protein,  in  the  form  of  casein,  supplies  the 
amino  acids  and  the  mineral  substances  are 
found  mostly  in  the  ash.  The  ferments  are  fur- 
nished by  the  digestive  organs  of  the  young.  Di- 
gestion is  relatively  simple  and  easy,  absorption 
proceeds  quickly  and  growth  follows. 

Bacteria,  although  unicellular  and  simple 
morphologically,  are  made  up  chemically  of 
highly  complex  molecules.  There  may  be  uni- 
cellular organisms  composed  of  simple  proteins 
but  this  certainly  is  not  true  of  the  bacteria 
which  I  have  studied.  In  their  chemical  com- 
position and  structure  these  bacteria  are  quite 
as  complex  as  the  most  highly  developed  cells 
in  the  animal  body.  It  follows,  therefore,  that 
when  we  speak  of  bacteria  as  low  and  primitive 
forms  of  life,  we  should  bear  in  mind  that  we 
are  speaking  as  morphologists  and  not  as  chem- 
ists. Many,  probably  all,  of  the  soluble  proteins 
in  man's  body  are  chemically  of  much  simpler 
structure  than  are  those  of  the  bacterial  cell. 

The  Proteoses. 

Schmidt-Mulheim  in  trying  to  discover  the 
fate  of  peptone  in  the  blood  (it  being  assumed 


THE    PROTEOSES  49 

at  that  time  that  peptones  are  absorbed  as  such 
into  the  blood)  found  that  the  intravenous  in- 
jection of  Witte's  peptone,  after  the  removal 
of  the  undigested  proteins  from  this  commer- 
cial preparation,  caused  in  dogs  striking  physi- 
ological effects.  The  most  notable  among  these 
were:  (1)  an  inhibiting  action  on  the  coagula- 
tion of  the  blood  and  (2)  a  rapid  and  marked 
reduction  in  blood  pressure.  This  work  done  in 
Ludwig's  laboratory  was  continued  a  year  later 
by  Fano.  Furthermore,  it  was  shown  that  a 
second  injection  of  peptone  made  shortly  after 
recovery  from  the  effects  of  the  first  had  but  lit- 
tle effect.  From  these  observations  it  became 
customary  to  speak  of  "peptone  poisoning" 
and  "peptone  innnunity.''  Fano  did  not  con- 
fine his  work  to  Witte's  peptone,  but  made  his 
own  product  by  the  digestion  of  fibrin  with  pep- 
sin and  trypsin.  He  also  used  Griiber's  prepa- 
ration and  one  from  America.  Grosjean  used 
propeptone  and  peptone  prepared  by  the 
method  of  Kiihne  and  Chittenden  and  found 
that  the  former  had  a  marked  effect,  especially 
when  employed  in  doses  of  more  than  0.15  g. 
per  kilo.  Arthus  and  Huber  employed  caseoses 
prepared  by  pancreatic  digestion.  Chittenden, 
Mendel  and  McDermott  and  later  Chittenden, 
Mendel  and  Henderson  produced  highly  poi- 


50  POISONOUS   PROTEINS 

sonous  bodies  by  breaking  up  proteins  with  a 
vegetable  ferment,  papain,  also  with  super- 
heated steam  and  dilute  acid  without  the  aid  of 
any  ferment.  Moreover,  they  found  that  all  the 
primary,  digestive  protein  derivatives  have 
more  or  less  marked  effect  upon  blood  coagu- 
lation and  blood  pressure.  Pick  and  Spiro  were 
unable  to  obtain  a  poisonous  derivative  from 
pure  proteins,  edestin  and  casein,  and  con- 
cluded that  the  poisonous  agent  present  in 
mixed  bodies  is  not  a  protein  at  all  but  an  en- 
zyme for  which  they  proposed  the  name  pepto- 
zyme.  According  to  their  view  this  in  the  pro- 
enzyme stage  is  widely  distributed  in  the  animal 
body,  since  they  found  the  poison  among  the 
cleavage  and  digestive  products  of  many  or- 
gans. It  might  get  mixed  with  the  protein  split 
products  in  digestion  with  an  animal  ferment, 
as  pepsin  or  trypsin,  or  it  exists  in  the  tissue 
or  protein  which  undergoes  digestion ;  but  take 
a  pure  protein  like  edestin  or  casein,  and  split 
it  with  acid  and  no  poisonous  body  results. 
They  claim  that  the  poison  never  results  from 
the  hydrolysis  of  proteins  with  alkali.  This  is 
interesting  in  view  of  the  fact  that  we  have 
found  cleavage  with  dilute  alkali  the  best  way 
of  obtaining  the  protein  poison. 

Underhill  has  shown  the  incorrectness  of  the 


THE   PROTEOSES  51 

claim  of  Pick  and  Spiro  and  demonstrated  that 
the  proteoses  are  in  and  of  themselves  poison- 
ous, when  administered  intravenously.  He  pre- 
pared the  poison  from  pure  proteins  by  cleav- 
age with  acids  and  showed  that  native  proteo- 
ses found  in  seeds  and  nuts,  wheat  embryo, 
hemp  seed  and  Brazil  nuts,  when  introduced 
into  animals  intravenously  induce  all  the  symp- 
toms formerly  known  as  those  of  peptone  poi- 
soning. 

Popielski  has  worked  with  a  body  which  he 
extracts  from  commercial  peptone  with  alco- 
hol. This  '  '  vasodilatin, "  as  he  calls  it,  has  the 
same  action  that  was  formerly  attributed  to 
peptone  and  notwithstanding  its  solubility  in  al- 
cohol it  gives  the  protein  color  tests,  at  least 
the  biuret  and  the  Millon. 

It  must  be  evident  that  the  behavior  of  my 
protein  poison  both  chemically  and  physiolog- 
ically, closely  resembles  that  of  the  proteoses. 
Some  proteoses,  at  least,  are  soluble  in  alcohol, 
and  as  has  been  said,  Popielski  extracts  his 
body  from  commercial  peptones  with  alcohol. 
The  protein  poison,  though  soluble  in  absolute 
alcohol,  gives  the  protein  reactions  and  is  a 
biuret  body;  some  proteoses  behave  in  a  simi- 
lar manner.  Edmunds  has  shown  that  the  pro- 
tein poison  lowers  blood  pressure  in  dogs,  just 


52  POISONOUS   PROTEINS 

as  the  "peptone  poison"  does.  Edmunds  did 
not  find  that  the  protein  poison  inhibits  the 
coagulation  of  Mood,  but  Underhill  has  recent- 
ly showed  that  it  has  this  effect,  when  used  in 
larger  doses  than  those  employed  by  Edmunds. 
Underhill  has  recently  compared  the  action  of 
the  protein  poison  with  that  of  the  proteoses" 
and  finds  that  the  resemblance  is  strong  both  in 
the  effect  upon  blood  pressure  and  coagulation, 
but  "Vaughan's  preparation  differs  from  the 
proteoses  in  that  it  produces  marked  symptoms 
or  even  death  in  the  rabbit  in  relatively  small 
doses."  The  rabbit  is  mentioned  here  because 
of  its  known  refractoriness  to  proteoses. 

It  seems  to  me  highly  probable  that  the  poi- 
sonous group  in  the  proteoses  is  the  protein 
poison  and  that  its  more  powerful  action  is  due 
to  the  fact  that  it  has  been  more  effectually 
stripped  of  those  groups  which  tend  to  neutral- 
ize its  effects.  It  is  present  in  every  true  pro- 
tein and  when  molecular  disruption  proceeds  up 
to  a  certain  point,  the  physiological  action  is  in- 
creased, beyond  that  point  if  is  decreased.  The 
protein  poison  kills  dogs,  as  shown  by  Under- 
hill, in  doses  in  which  the  proteoses  have  only  a 
temporary  effect,  but  the  symptoms  are  the 
same.  From  this  I  conclude  that  the  poisonous 
group  is  the  same  in  both  instances,  but  the  free 


CLEAVAGE    OF   PROTEINS  53 

poison  is  more  effective  than  the  combined. 
This  belief  is  confirmed  by  the  fact  that  the 
free  poison  is  easily  split  out  of  the  proteoses 
by  proper  chemical  agents. 

The  Autolytic  Cleavage  of  Proteins. 

All  proteins  sooner  or  later  undergo  autoly- 
tic cleavage.  When  a  solution  or  suspension  of 
protein  in  water  or  salt  solution  is  protected 
from  bacterial  invasion  by  chloroform  or  toluol 
and  kept  at  about  37°  the  protein  undergoes 
spontaneous  cleavage.  SalkowsM  seems  to 
have  been  the  first  to  investigate  this  phenome- 
non scientifically.  This  work  has  been  contin- 
ued by  Biondi,  Schwiening,  Launoy,  Jacobi,  and 
others.  Most  of  these  have  given  attention  to 
cellular  autolysis,  as  this  is  the  most  interest- 
ing phase  of  the  subject,  but  all  proteins, 
whether  cellular  or  without  structure,  go 
through  a  similar  process.  Fibrin  undergoes 
autolysis  quite  as  promptly  as  liver  cells  do.  It 
is  well  known  that  in  multicellular  animals  pro- 
teases are  generally  distributed.  At  first  it  was 
assumed  that  these  consist  of  the  alimentary 
ferments  which  have  been  absorbed  and  distrib- 
uted through  the  body.  However,  research  has 
shown  that  the  autolytic  ferments  differ  from 
either  pepsin  or  trypsin.    In  the  first  place  they 


54  POISONOUS   PROTEINS 

are  possessed  of  a  degree  of  specificity  not  char- 
acteristic of  the  alimentary  enzymes.  The  fer- 
ment found  in  each  organ  or  each  kind  of  tis- 
sue digests  especially,  more  rapidly  and  com- 
pletely, the  organ  or  tissue  in  which  it  is  found. 
The  liver  ferment  readily  splits  up  liver  tissue 
but  is  less  effective  in  its  action  on  the  proteins 
of  other  organs.  In  the  second  place,  the  prod- 
ucts of  autolytic  cleavage  differ  from  those  o'f 
enteral  digestion.  Pepsin  forms  large  amounts 
of  primary  cleavage  products,  such  as  proteo- 
ses and  peptones.  These,  especially  the  former, 
are  highly  poisonous,  and  would  have  a  most 
disastrous  effect  were  they  liberated  parenter- 
ally.  The  autolytic  enzymes  produce  none  or 
only  traces  of  these  primary  split  products. 
They  cleave  deeper  and  their  chief  products  are 
the  relatively  harmless  amino  acids  and  purin 
bodies.  From  tryptic.  digestion  the  autolytic 
enzymes  differ  in  several  particulars.  Trypsin 
acts  in  feebly  alkaline  solution  while  autolysis 
proceeds  most  rapidly  in  slightly  acid  media.  It 
is  more  than  probable  that  the  intracellular  tis- 
sue is  always  feebly  acid.  Tryptophan,  a  prod- 
uct of  tryptic  digestion,  is  seldom  or  never 
found  among  the  autolytic  products.  In  auto- 
lytic cleavage  of  proteins  much  more  ammonia 
is  found  than  in  tryptic  digestion.     Further- 


CLEAVAGE   OF   PROTEINS  55 

more  the  autolytic  enzymes  persist  in  animals 
from  which  the  pancreas  has  been  removed.  We 
see  from  these  facts  that  protein  tissues  disinte- 
grate normally  in  the  animal  body  without  the 
formation  of  poisonous  products.  It  must  be 
admitted  that  in  certain  pathological  conditions, 
such  as  acute  yellow  atrophy  of  the  liver  and  in 
phosphorus  poisoning,  autolysis  proceeds  with 
harmful  rapidity  and  becomes  at  least  a  highly 
destructive  process. 

It  has  been  suggested  that  the  autolytic  en- 
zymes are  constituents  of  the  blood  and  are 
generally  distributed  through  the  body  by  this 
fluid.  In  other  words  it  has  been  held  that  they 
are  blood  ferments.  That  this  is  not  true  is 
shown  by  the  fact  that  blood  and  blood  serum 
have  an  inhibiting  effect  upon  autolytic  action. 
Besides,  proteins  which  contain  no  blood,  such 
as  egg-white,  undergo  autolytic  cleavage. 

The  study  of  autolytic  cleavage  is  compli- 
cated by  the  presence  in  many  proteins  of  other 
ferments  such  as  nucleases,  arginases,  etc. 
What  effects  the  autolytic  enzymes  have  upon 
foreign  proteins  is  a  question  of  importance, 
but  one  which  cannot  be  answered  at  present. 
It  will  be  understood  that  I  have  been  speak- 
ing so  far  of  the  autolytic  enzymes  of  the  cellu- 
lar and  other  proteins  of  the  multicellular  ani- 


56  POISONOUS   PKOTEINS 

mal.  When  we  come  to  speak  of  the  autolytic 
cleavage  of  unicellular  organisms,  snch  as  bac- 
teria, we  have  quite  a  different  problem.  That 
bacteria  undergo  autolytic  cleavage  and  that 
the  products  formed  in  this  process  may  be 
harmful  to  multicellular  organisms  has  been 
abundantly  shown.  Old  cultures  of  colon  and 
typhoid  bacilli  may  contain  soluble  split  prod- 
ucts which  are  highly  harmful  and  indeed  may 
be  fatally  effective  in  their  action  on  the  higher 
animals.  Whether  pathogenic  bacteria  undergo 
autolytic  cleavage  in  the  bodies  of  their  hosts  is 
a  question  which,  so  far  as  I  know,  has  not  been 
decisively  determined  by  experiment.  The  pre- 
sumption is  that  this  may  and  does  happen. 

The  following  experimental  data  concerning 
the  autolytic  cleavage  of  bacterial  proteins  may 
be  of  interest  in  this  connection : 

Eosenow  has  shown  that  pneumococci  sus- 
pended in  salt  solution  and  kept  at  37°  for  forty- 
eight  hours,  under  ether  or  over  chloroform, 
undergo  autolysis  with  the  liberation  of  a  poi- 
son. This  poison  injected  intravenously  or  in- 
tracardiacly  in  normal  animals  induces  ana- 
phylactic shock.  In  guinea-pigs  death  results 
from  bronchial  spasm  and  consequent  arrest  of 
respiration.    In  dogs  it  causes  marked  fall  in 


CLEAVAGE    OF   PEOTEIXS  57 

blood  pressure  and  delays  the  coagulation  of 
the  blood. 

I  took  powdered  pneumococcus  cellular  sub- 
stance which  had  been  prepared  nearly  seven 
years  before.  Microscopic  examination  showed 
the  pneumococci  as  clearly  denned  and  in  as 
perfect  form  as  nTa  fresh  preparation.  Five 
hundred  milligrams  of  this  powder  was  sus- 
pended in  500  c.c.  of  salt  solution,  10  c.c.  of 
chloroform  added  and  kept  at  37°.  After 
twenty-four  hours  10  c.c.  of  the  opalescent  su- 
pernant  fluid  was  administered  to  a  guinea-pig 
intravenously.  "Within  two  hours  the  animal's 
temperature  fell  to  94°,  but  recovery  followed. 
The  same  experiment  repeated  after  48  and  72 
hours  killed  the  animal  within  two  hours  with 
the  symptoms  of  subacute  anaphylactic  shock. 
A  like  injection  after  six  days  killed  within 
three  minutes  with  all  the  symptoms  and  post- 
mortem findings  of  acute  anaphylactic  shock. 

It  has  been  shown  that  the  cholera  bacillus 
does  not  undergo  ready  autolytic  cleavage  in 
vitro,  but  there  is  reason  for  suspecting  that 
this  happens  in  the  intestine  of  infected  men, 
since  after  death  the  bacillus  is  found  only  in 
the  intestinal  canal,  in  some  instances  at  least, 
all  the  internal  organs  being  sterile. 

Warden  finds  that  the  gonococcus  early  un- 


58  POISONOUS   PROTEINS 

dergoes  autolysis  and  that  the  autolysates  are 
fatal  to  guinea-pigs.  He  believes  that  the  au- 
tolysis of  this  organism  is  not  due  to  enzyme, 
hut  results  from  a  disruption  caused  by  the  ab- 
sorption of  water  by  the  cells. 

Parenteral  Protein  Digestion. 

IVe  now  distinguish  between  enteral  and  pa- 
renteral digestion.  We  take  diverse  proteins 
into  our  alimentary  canals  and  through  the  ac- 
tivity of  the  enteral  digestive  ferments  they  are 
split  into  amino  acids  which  are  utilized  by  the 
body  cells  in  growth  and  in  function.  This  is 
the  normal  way  in  which  the  body  cells  of  the 
higher  animals  are  fed,  for  the  most  part  at 
least.  Under  normal  conditions  the  amount  of 
protein  reaching  the  blood  and  lymph  undi- 
gested is  small  and  negligible  in  effect.  Minute 
bits  of  unbroken  protein  may  find  their  way  into 
the  circulation  through  the  respiratory  and  di- 
gestive tracts.  These,  entering  through  the 
respiratory  organs,  may  cause  local  sensitiza- 
tion which  manifests  itself  in  the  complex  of 
symptoms  usually  designated  as  hay — rose — or 
horse-fever  and  asthma.  Those  passing  in  un- 
digested forms  through  the  walls  of  the  ali- 
mentary canal  may  lead  to  the  untoward  ef- 
fects of  certain  articles  of  diet  and  possibly 


PARENTERAL   DIGESTION  59 

may  exert  a  more  serious  action  on  some  of  the 
more  distant  organs,  especially  the  kidneys. 

During  fetal  life  all  the  food  enters  the  body 
parenterally  and  there  is  no  enteral  digestion. 
There  are  reasons  for  suspecting  that  during 
infancy  the  chief  milk  protein,  casein,  may  be 
absorbed  in  part  in  an  unbroken  state.  At  least 
in  a  few  instances  unchanged  casein  has  been 
detected  by  the  biological  test  in  the  blood  of  in- 
fants suffering  from  summer  diarrhea. 

In  my  opinion  there  are  reasons  for  believing 
that  in  some  animals  a  certain  part,  or  certain 
kinds,  of  protein  food  are  absorbed  unbroken 
and  are  digested  parenterally.  Babbits  are 
easily  sensitized,  notably  by  casein  fed  by  the 
mouth  or  administered  by  the  rectum.  I  have 
detected  the  protein  in  the  heart's  blood  by  the 
biological  test  after  such  feedings.  While  there 
is  a  promising  field  for  research  along  these 
lines,  it  is  safe  to  say  that  in  man  in  health,  the 
amount  of  unbroken,  foreign  protein  reaching 
the  circulation  is  small.  Protein  in  appreciable 
quantities  reaches  the  blood  only  when  injected, 
as  in  the  employment  of  sera  and  vaccines  or 
through  infection.  In  the  latter  instance  the 
protein  multiplies  in  the  body. 

It  is  evident  that  one  or  more  of  the  follow- 
ing effects  may  result  from  the  parental  intro- 


60  POISONOUS   PROTEINS 

duction  of  a  foreign  protein.  (1)  It  may  be  elim- 
inated through  the  kidneys.  (2)  It  may  be 
passed  into  the  alimentary  canal  and  there  di- 
gested. (3)  It  may  be  digested  parenterally. 
All  these  dispositions  may  be  employed  in  the 
disposal  of  the  foreign  protein. 

The  literature  concerning  the  renal  elimina- 
tion of  foreign  proteins  is  voluminous,  but  often 
contradictory.  The  occurrence  and  extent  of 
this  form  of  disposal  vary  with  the  kind  of  pro- 
tein, the  quantity,  the  rapidity  of  introduction, 
the  species  and  individuality  of  the  animal  and 
probably  upon  many  unknown  conditions.  It 
was  formerly  supposed  that  all  the  protein  pass- 
ing through  the  kidneys  after  parenteral  intro- 
duction consists  of  that  introduced.  It  has  been 
definitely  shown  that  this  is  not  true  and  the  es- 
timates found  in  the  older  literature  showing 
the  per  cent  eliminated  by  the  kidney  are  with- 
out value.  Some  years  ago  it  was  shown  in  my 
laboratory  that  in  the  urine  of  rabbits  after  the 
parenteral  introduction  of  egg-white,  both  egg- 
white  and  blood  protein  appear.  Guinea-pigs 
were  sensitized  to  both  with  the  urine.  This 
gives  no  indication  of  the  proportion  in  which 
they  were  present.  It  has  been  shown  by  Chi- 
ray  and  confirmed  in  my  laboratory  that  for- 
eign protein  injected  into  the  blood  soon  dis- 


PARENTERAL   DIGESTION  61 

appears  from  the  circulating  fluid  and  carries 
with  it  an  appreciable  amount  of  the  proteins  of 
the  blood.  So  far  as  I  know  Chiray  is  the  only 
one  who  has  made  frequent  observations  of  the 
effects  of  the  parenteral  administration  of  pro- 
teins in  man.  He  frequently  induced  albumi- 
nuria in  this  way,  especially  in  those  who  al- 
ready showed  renal  inefficiency.  In  rabbits  he 
induced  marked  structural  changes  in  the  kid- 
neys by  repeated  injections. 

In  my  work  on  the  parenteral  introduction  of 
proteins,  I  have  carefully  controlled  the  rate  of 
injection  and  have  found  that  the  foreign  pro- 
tein is  more  likely  to  appear  in  the  urine  when 
the  rate  of  injecton  is  high.  When  the  protein 
is  slowly  introduced,  I  have  been  surprised  at 
the  large  amount  that  can  be  introduced  into 
the  abdominal  cavity  or  into  an  ear  vein  with- 
out any  detectable  trace  appearing  in  the  urine. 

When  heterologous  proteins  are  injected  into 
the  blood  they  soon  find  their  way  into  the  in- 
testinal lumen.  They  are  poured  in  with  the 
bile  and  they  pass  into  the  abdominal  cavity  and 
through  the  intestinal  walls.  With  the  biolog- 
ical test  we  have  detected  proteins  injected  into 
the  ear  veins  of  rabbits  in  the  liver,  abdominal 
cavity  and  lumen  of  the  intestines.  It  seems  to 
be  a  general  physiological  law  that  poisons  in- 


62  POISONOUS   PROTEINS 

troduced  into  the  blood  are  eliminated  in  part 
at  least  into  the  alimentary  canal.  Morphine 
given  subcutaneously  may  be  detected  in  wash- 
ings from  the  stomach.  Gastric  erosion  may  be 
induced  by  the  subcutaneous  or  intravenous  ad- 
ministration of  arsenical  preparations.  So 
long  ago  as  1753  Sproegel  showed  that  gastric 
lesions  may  be  due  to  arsenic  absorbed  from 
wounds,  and  since  that  time  they  have  been  in- 
duced in  animals  by  the  hypodermic  adminis- 
tration of  neutral  solutions  of  arsenic.  Similar 
lesions  are  seen  in  poisoning  with  antimony  and 
other  metals  and  may  result  in  these  instances 
also  from  application  made  to  wounds  and  to 
raw  surfaces.  Mercury  when  employed  by  in- 
unction is  poured  into  the  alimentary  canal  and 
its  destructive  action  may  be  seen  in  almost  any 
part  from  the  mouth  to  the  rectum.  Erosions 
of  the  stomach  and  intestine  may  be  extensive 
and  deep,  even  to  perforation.  The  fact  that 
gastric  and  duodenal  ulcers  may  follow  severe 
burns  of  the  skin  has  been  long  known  and  is 
best  explained  by  supposing  them  due  to  the 
large  amount  of  poison  resulting  from  the  burn, 
being  brought  to  the  walls  of  the  alimentary 
canal.  The  gastric  inflammations  and  erosions 
of  the  acute  infectious  diseases  are  doubtlessly 
due  to  the  same  cause.    The  smallpox  virus  has 


PAEENTEEAL   DIGESTION  63 

a  predilection  for  epithelial  tissues  and  mani- 
fests its  destructive  action  in  the  skin  and  in 
mucous  membranes.  It  has  long  been  known 
that  peptic  ulcer  is  frequently  associated  with 
chronic  appendicitis  and  the  recent  brilliant 
work  of  Eosenow  has  called  attention  to  the 
probable  relation  between  peptic  ulcer  and 
pyorrhea.  In  case  of  a  nidus  of  infection  in 
any  part  of  the  body  poisonous  proteins  are  be- 
ing poured  into  the  circulation  and  these  like 
other  poisons  are  carried  to  the  walls  of  the  in- 
testine for  the  evident  purpose  of  elimination. 
Here  they  accumulate  and  in  their  reaction  with 
the  body  cells,  the  latter  are  more  or  less  in- 
jured. The  elimination  of  proteins  from  the 
blood  into  the  alimentary  canal  holds  for  both 
living  and  dead,  formed  and  unformed  proteins. 
This  is  an  interesting  phase  in  the  study  of  the 
action  of  poisonous  proteins  and  is  worthy  of 
further  study. 

It  has  been  long  known  that  blood  serum,  like 
living  cells,  is  highly  resistant  to  proteolytic 
enzymes.  Furthermore,  the  presence  of  blood 
serum  markedly  retards  both  peptic  and  pan- 
creatic digestion.  It  has  been  generally  in- 
ferred from  these  facts  that  blood  serum  con- 
tains an  antiproteolytic  ferment  and  since  the 
reaction  is  alkaline,  this  is  generally  designated 


64  POISONOUS   PROTEINS 

as  antitrypsin.  So  far  as  I  know,  Camus  and 
Gley  were  the  first  to  show  experimentally  that 
blood  serum  inhibits  peptic  and  tryptic  action. 
These  investigators  observed  that  fibrin  or  co- 
agulated egg-white  placed  in  serum  and  treated 
with  active  pepsin  or  trypsin  remains  intact. 
More  extended  observations  have  shown  that 
many,  if  not  all,  kinds  of  proteolytic  digestion, 
are  retarded,  often  wholly  arrested,  by  the  pres- 
ence of  blood  serum.  There  is  another  inter- 
esting fact  in  this  connection.  The  injection  of 
proteolytic  ferments  into  an  animal,  especially 
repeated  injections,  increases  the  potency  of  the 
blood  serum  in  the  inhibition  of  the  action  of 
that  ferment.  Antibodies  are  formed  and  ac- 
cumulate in  the  blood  after  repeated  injections 
of  pepsin,  trypsin,  rennin,  etc.  The  effect  of 
such  injections  is  similar,  probably  closely  re- 
lated, to  that  which  follows  injections  of  toxins. 
But  little  is  known  concerning  these  antibodies 
in  case  of  either  the  ferments  or  the,  toxin. 

Delezenne  and  Pozerski  first  showed  that 
chloroform  removes  from  blood  serum  the  anti- 
proteolytic  body.  'They  found  that  blood  serum 
has  no  digestive  action  on  gelatin  under  ordi- 
nary conditions,  but  that  blood  serum  which  has 
been  extracted  with  chloroform  promptly  di- 
gests gelatin.     The  researches  of  Jobling  and 


PARENTERAL   DIGESTION  65 

others  have  confirmed  and  amplified  this  work 
and  it  has  been  shown  that  when  the  unsatu- 
rated fatty  acids  are  removed  from  blood  se- 
rum by  extraction  with  chloroform  or  ether,  it 
becomes  highly  poisonous  even  for  the  species 
from  which  it  was  derived.  Whether  Jobling  is 
right  in  his  contention  that  the  fatty  acids  con- 
stitute the  antibody  is  still  to  be  determined.  It 
is  possible  that  the  extraction  of  blood  serum 
with  chloroform  may  have  some  effect  upon  the 
equilibrium  in  its  protein  constituents. 

Friedberger  found  that  the  blood  serum,  of 
the  guinea-pig  when  incubated  with  bacterial 
cell  substance  becomes  poisonous.  He  ex- 
plained this  on  the  assumption  that  the  pro- 
teases of  the  serum  digest  the  bacterial  cells 
with  the  formation  of  a  poison  which  he  calls 
anaphylatoxin.  Later  it  was  shown  that  the 
guinea-pig  serum  when  incubated  with  agar  or 
starch  becomes  poisonous.  From  these  find- 
ings it  was  suspected  that  bacillary  substances, 
agar  and  starch,  act  upon  guinea-pigs  by  ab- 
sorption of  the  antibodies.  In  this  way  the  pro- 
teases in  the  serum  are  relieved  of  the  presence 
of  their  antibodies  and  digest  the  proteins  in 
the  serum.  In  other  words,  the  matrix  of  the 
poison  consists  of  the  proteins  in  the  serum 
and  not  of  the  bacillary  cell  substance. 


66  POISONOUS   PROTEINS 

Abderhalden  found  that  when  placental  tis- 
sue is  digested  with  the  serum  of  pregnant 
women  diffusible  digestive  products  are  formed 
and  may  be  detected  in  the  diffusate  by  the 
biuret  and  ninhydrin  tests.  He  explained  this  by 
supposing  that  placental  tissue  in  small  amount 
finds  its  way  into  the  maternal  blood,  and  that 
this  fluid  acquires  the  property  of  digesting 
placental  proteins.  Abderhalden  believes  this 
to  be  a  specific  reaction  and  has  proposed  it  as 
a  diagnostic  test  for  pregnancy.  This  test  has 
been  studied  by  many  and  while  its  significance 
cannot  be  considered  as  finally  settled  the 
weight  of  evidence  seems  to  be  that  Abderhal- 
den's  explanation  is  not  correct.  It  seems  from 
the  evidence  now  at  hand  that  the  placental  tis- 
sue absorbs  the  antiferments  and  the  unop- 
posed protease  of  the  serum  digests  the  protein 
constituents  of  this  fluid. 

The  weight  of  evidence  today  discards  the 
idea  of  specific  proteases  in  blood  serum  and  fa- 
vors the  idea  that  certain  antibodies  exist  in  the 
serum  and  when  these  are  reduced  in  amount, 
the  nonspecific  protease  of  the  blood  serum  acts 
upon  its  own  protein  constituents.  It  must  be 
admitted  that  this  view  is  more  in  accordance 
with  some  of  the  facts  than  the  one  which  holds 
that  specific  proteases  are  existent  in  the  blood 


PARENTERAL   DIGESTION  67 

or  may  be  brought  into  existence.  However,  it 
should  be  stated  that  the  present  view  does  not 
exclude  the  necessity  of  regarding  protein  di- 
gestion in  the  blood  as,  in  some  instances  at 
least,  specific.  Take  the  production  of  anaphy- 
lactic shock  as  an  example.  The  theory  pro- 
posed by  "Wheeler  and  me  in  1907  supposes  that 
when  a  given  protein  is  first  injected  par  enter- 
ally  into  an  animal,  it  slowly  develops  a  specific 
protease.  This  is  a  cellular  product.  Certain 
cells  stimulated  by  contact  and  by  penetration 
with  the  foreign  protein  develop  a  new,  specific 
protease  which  is  capable  of  digesting  that  pro- 
tein and  no  other.  The  protein  of  the  first  in- 
jection is  disposed  of  by  this  new  specific  fer- 
ment, but  is  broken  up  so  slowly  that  no  harm 
comes  to  the  animal,  or  at  least  no  recognizable 
danger,  from  the  cleavage  products.  The  cells 
continue  in  the  possession  of  the  newly  acquired 
function.  This  may  persist  for  years  and  in- 
deed throughout  life.  The  animal  is  said  to  be 
sensitized.  On  reinjection  of  the  same  protein 
the  body  cells,  having  acquired  the  function  of 
digesting  it,  do  so  with  such  violence  that  the 
digestive  products  endanger  the  life  of  the  ani- 
mal or  at  least  develop  physiological  disturb- 
ances which  are  easily  recognizable.  "We  have 
offered  this  in  explanation  of  the  success  of  vac- 


bb  POISONOUS   PROTEINS 

cination.  The  vaccine  virus  is  introduced  into 
the  child's  arm.  The  proteins  of  which  the  vi- 
rus is  composed  are  distributed  in  the  body  and 
sensitize  certain  cells.  This  means  that  the 
cells  develop  a  ferment  which  destroys  the  vac- 
cine virus  and  the  new  function  developed  in 
these  cells  by  their  first  experience  with  the 
smallpox  protein  in  its  attenuated  form  con- 
tinues in  the  possession  of  the  cells  for  years. 
"When  the  vaccinated  person  is  exposed  to  small- 
pox the  virus  of  the  disease  is  destroyed  before 
it  has  time  to  multiply  and  consequently  the  in- 
dividual is  protected  from  the  disease.  Please 
understand  that  I  am  not  ready  to  give  up 
the  theory  of  the  formation  of  specific  pro- 
teases. I  see  no  other  explanation  of  the  im- 
munity conferred  by  vaccination  or  by  one  at- 
tack of  the  disease.  However,  in  presenting 
this  matter  I  wish  to  proceed  without  being  in- 
fluenced by  preconceived  ideas,  and  I  wish  to 
repeat  that  the  idea  of  a  nonspecific  protein  di- 
gestion in  anaphylactic  shock  especially  has 
much  in  its  favor,  both  in  fact  and  in  theory. 
The  poison  developed  in  anaphylactic  shock 
may  not  come  from  the  protein  of  the  reinjec- 
tion  and  the  protease  developed  in  sensitiza- 
tion may  not  be  specific.  Anaphylactic  shock 
may  be  due  wholly  to  the  unmasking  of  a  non- 


PAKENTEEAL   DIGESTION  69 

specific  ferment  and  the  poison  formed  may 
come  from  the  proteins  of  the  blood,  but  if  all 
this  be  true,  and  the  weight  of  evidence  today 
is  in  this  direction,  the  anaphylactic  reaction 
remains  specific.  We  have  only  transferred  the 
problem  of  specificity  from  the  development 
of  a  specific  enzyme  to  the  specific  uncovering 
of  a  nonspecific  enzyme.  It  remains  true  that 
an  animal  sensitized  to  one  protein  is  not  sen- 
sitized to  other  and  unlike  proteins. 

I  have  said  that  the  theory  of  the  uncovering 
of  a  general  protease  in  anaphylactic  shock  has 
much  in  its  favor.  The  blood  seems  to  be  a 
fluid  in  which  ferments  and  antiferments  are 
nicely  and  delicately  balanced  and  a  slight  dis- 
turbance in  this  equilibrium  leads  to  marked 
effect.  We  have  obtained  from  one  gram  of 
casein  enough  of  the  protein  poison  to  kill  800 
guinea-pigs  when  injected  intravenously.  That 
casein,  the  chief  protein  constituent  of  the  food 
of  all  mammalian  young,  should  be  found  to 
contain  a  body  so  highly  poisonous  when  intro- 
duced intravenously  is  certainly  a  surprising 
thing.  However,  the  surprise  does  not  disap- 
pear when  we  go  further  and  find  that  a  simi- 
lar poison  may  be  obtained  not  only  from  all  the 
proteins  we  eat  but  also  from  those  that  make 
up  the  tissue  of  our  own  bodies.    Indeed,  every 


70  POISONOUS   PEOTEINS 

gram  of  protein  in  an  animal's  body  may  sup- 
ply enough  poison  to  kill  many  such  animals. 
There  are  other  interesting  things  about  this 
protein  poison  besides  its  potency.  When 
amounts  of  it,  even  smaller  than  the  minimum 
lethal  dose,  are  incubated  with  blood  serum  in 
vitro,  the  serum,  in  itself  inert,  becomes  fatally 
poisonous.  In  these  studies  a  curious  phenom- 
enon has  been  observed.  The  incubating  serum 
containing  the  poison  may  be  fatally  active  at 
the  expiration  of  a  given  time,  then  later  wholly 
without  effect,  and  later  still  fatally  active. 
This  wave  of  appearing,  disappearing,  reap- 
pearing toxicity  we  have  frequently  observed. 
For  it,  I  have  not  even  the  shadow  of  an  expla- 
nation. It  may  turn  out  after  all  that  ferments 
and  antiferments  are  not  concerned  in  these 
phenomena.  I  have  tried  to  think  of  oscillations 
induced  in  a  colloidal  fluid  like  the  blood  serum 
by  the  presence  of  the  protein  poison,  but  I  have 
not  been  able  to  fix  such  a  concept. 

As  was  first  shown  by  Friedberger  bacterial 
cellular  substance  incubated  with  blood  serum 
in  vitro  renders  the  serum  poisonous.  In  re- 
peating these  experiments  and  injecting  the  se- 
rum at  intervals,  at  one  time  it  kills  with  all  the 
violence  of  anaphylactic  shock,  then  it  has  no 
effect,  then  again  it  kills.    I  have  tried  to  time 


PARENTERAL   DIGESTION  71 

this  wave  of  toxicity,  but  adjust  every  condition 
to  the  best  of  my  ability,  I  have  been  unable  to 
chart  it.  It  is  to  be  hoped  that  some  wiser  man 
with  more  perfect  control  of  the  conditions  of 
his  experiments  will  solve  this  question.  I  am 
willing  to  leave  it  to  those  braver  than  I  to 
try  on  human  beings  such  poisonous  mixtures 
of  bacterial  proteins  as  phylacogen. 

I  have  stated  that  I  am  not  yet  ready  to  give 
up  the  idea  that  the  parenteral  introduction  of 
foreign  proteins  produces  specific  alterations  in 
the  blood.  I  cannot  do  so,  so  long  as  I  have  the 
evidence  supplied  by  the  specificity  of  agglu- 
tination and  precipitin  reactions.  We  may 
have  no  proof  that  these  are  due  to  the  develop- 
ment of  specific  proteases,  but  whatever  their 
action  it  is  within  certain  limits  specific.  Some 
years  ago  with  my  assistants  I  published  the 
results  of  work  which  I  interpreted  as  demon- 
strating the  elaboration  of  specific  proteases  in 
sensitized  animals.  The  results  were  all  so 
clean  cut  and  uniform  that  they  were  convinc- 
ing to  me  at  least.  I  will  give  a  brief  abstract : 
(1)  One  milligram  of  egg-white  incubated  at 
37°,  for  thirty  minutes  in  5  c.c.  of  the  serum  or 
organ  extract  of  unsensitized  guinea-pigs  is 
without  effect  when  injected  into  the  heart  of 
another  unsensitized  guinea-pig.     (2)  Like  re- 


72  POISONOUS   PROTEINS 

suits  followed  when  the  incubation  was  made 
with  fluids  obtained  from  an  animal  sensitized 
three  days  previously  with  egg-white.  (3) 
When  the  fluids  were  obtained  from  animals 
sensitized  fourteen  days  previously  to  egg- 
white,  anaphylactic  shock  followed  in  all.  (4) 
With  the  conditions  as  in  (3)  except  that  the  in- 
cubation was  prolonged  to  ninety  minutes,  the 
effects  were  less  marked.  (5)  With  the  condi- 
tions the  same  as  in  (3)  except  that  the  incuba- 
tion was  done  in  a  cold  room  the  effects  were 
nil.  (6)  "When  the  fluids  were  obtained  from  an 
animal  seventeen  days  after  sensitization,  ana- 
phylactic shock  resulted.  (7)  Filtration  of  the 
fluids  through  hard  fiber  paper  did  not  affect 
the  results.  (8)  Filtration  of  the  serum  and  or- 
gan extract  through  a  Berkefeld  V  did  not  af- 
fect the  results.  (9)  Filtration  after  incuba- 
tion did  not  affect  the  results.  (10)  When  the 
serum  and  organ  extracts  were  heated  to  56° 
for  thirty  minutes  there  were  no  effects.  (11) 
When  the  heated  serum  and  organ  extract  were 
activated  by  the  addition  of  unheated  serum 
and  extracts  the  effects  were  positive.  (12) 
When  the  serum  and  organ  extracts  of  a 
guinea-pig  sensitized  to  egg-white  were  incu- 
bated with  horse  serum  there  were  no  effects. 
(13)  The  serum  and  organ  extracts  of  guinea- 


PAKESTTEEAL   DIGESTION  73 

pigs  sensitized  to  horse  serum  elaborated  a 
poison  when  incubated  with  horse  serum.  (14) 
The  serum  and  organ  extracts  of  gninea-pigs 
sensitized  to  typhoid  bacilli  gave  a  poison  when 
incubated  with  typhoid  bacilli.  (15)  Like  re- 
sults were  obtained  with  the  cholera  bacillus. 

(16)  In  case  of  egg-white  the  serum  ceases  to  be 
active  in  about  forty  days  after  sensitization. 

(17)  When  the  amount  of  protein  incubated 
with  the  serum  and  organ  extracts  was  larger 
than  1  mg.  per  5  c.c.  the  results  were  less  cer- 
tain. 

If  there  was  not  more  luck  than  science  in 
these  experiments  they  clearly  show  specificity. 
I  now  know  that  there  would  be  a  chance  of  get- 
ting some  positive  results  with  a  nonspecific  se- 
rum, but  it  seems  impossible  for  these  results 
to  have  been  so  uniform  on  any  other  ground 
than  that  of  specificity.  Besides,  they  compare 
with  the  results  obtained  by  Pf eifTer  who  found 
that  the  sera  of  guinea-pigs  digested,  for  about 
forty  days  after  sensitization,  the  protein  to 
which  the  animal  had  been  sensitized.    / 

Abderhalden  and  his  students  in  numerous 
experiments  have  shown  by  the  polariscope  that 
the  blood  serum  of  a  sensitized  animal  has  a 
more  marked  digestive  action  on  the  specific 
anaphylactogen  than  has  the  serum  of  a  non- 


74  POISONOUS   PROTEINS 

sensitized  animal.  Similar  results  have  been 
obtained  by  dialysis  methods  by  PfeifTer  and 
Mita,  by  Pf  eifTer  and  Jarisch,  and  by  Znnz  and 
Gyorgy.  The  last  mentioned  have  apparently 
shown  a  marked  increase  in  amino  acids  during 
anaphylactic  shock.  This  controverts  the  find- 
ing of  Aner  and  Van  Slyke. 

I  have  repeatedly  found,  as  others  have,  that 
the  blood  serum  shows  sensitization  for  rela- 
tively a  short  time,  while  the  animal  remains  in 
a  sensitized  condition  much  longer.  This  ob- 
servation has  convinced  me  that  protein  sensiti- 
zation is  accompanied  by  and  is  due,  in  some  in- 
stances at  least,  to  a  profound  and  lasting  im- 
pression made  on  the  cells  of  the  body.  Indeed, 
there  can  be  no  doubt  that  protein  sensitization 
is  cellular.  Pearce  and  Eisenbrey  bled  a  sensi- 
tized dog  into  a  fresh  one  and  at  the  same  time 
replaced  the  blood  taken  from  the  sensitized 
one  by  that  of  a  second  fresh  one.  The  sensi- 
tized dog  from  which  all  its  blood  had  been  re- 
moved responded  with  anaphylactic  shock  on 
reinjection,  while  the  dog  now  carrying  all  the 
blood  of  the  sensitized  one  did  not. 


PAET  III. 

PROTEIN  FEVER, 

It  has  been  known  for  a  long  time  that  the 
parenteral  introduction  of  proteins  in  the  ani- 
mal body  may  be  followed  by  fever.  As  early 
as  1883  Roques  collected  the  literature  of  this 
subject  and  reported  his  own  experimental 
studies.  A  few  years  later  G-amaleia  made  a 
most  important  contribution  to  this  subject. 
The  title  of  this  paper  is  significant  and  reads 
as  follows:  "The  Destruction  of  Bacteria  in 
the  Febrile  Organism/ '  Gamaleia  found  that 
fever  follows  the  parenteral  introduction  of  bac- 
terial protein,  both  pathogenic  and  nonpatho- 
genic, both  living  and  dead,  consequently  he 
concluded  that  fever  is  a  result  not  directly  of 
bacterial  growth,  but  of  bacterial  destruction  in 
the  body.  Indeed,  he  observed  that  attenuated 
bacteria  often  induce  a  higher  and  more  per- 
sistent fever  than  the  virulent  forms.  When  a 
rabbit  is  inoculated  with  a  virulent  anthrax  ba- 
cillus, fever  develops  but  persists  only  a  few 
hours,  and  then  the  temperature  falls  below  the 
normal  and  death  occurs.    On  the  other  hand, 


76  POISONOUS   PROTEINS 

when  the  second  vaccine  is  used  on  a  fresh  ani- 
mal, fever  appears  and  continues  for  three 
days.  When  a  highly  virulent  anthrax  bacillus 
is  employed  there  may  be  no  fever  and  death 
follows  within  six  or  seven  hours.  Gamaleia 
made  similar  observations  in  other  infections 
and  came  to  the  following  conclusion :  ' i  Fever 
is  not  a  result  of  bacterial  growth,  but  on  the 
contrary  is  consequent  upon  a  reaction  on  the 
part  of  the  body  against  the  bacteria  and  leads 
to  their  destruction. ' '  Furthermore  he  found 
that  nonpathogenic  bacteria,  living  or  dead, 
led  to  the  development  of  fever.  I  think  that 
these  experiments,  made  more  than  a  quarter 
of  a  century  ago,  furnish  strong  support  of  my 
theory  that  fever  is  due  to  the  parenteral  de- 
struction of  proteins.  One  year  later  this  work 
was  confirmed  by  Charrin  and  Buffer  and  was 
shown  to  hold  good  for  nonbacterial  proteins  as 
well.  In  1890  Buchner  induced  the  characteris- 
tic phenomena  of  inflammation — calor,  rubor, 
tumor,  and  dolor — by  the  subcutaneous  injec- 
tion of  diverse  bacterial  proteins.  Krehl  and 
Matthes  induced  fever  by  the  parenteral  ad- 
ministration of  albumoses  and  peptones,  but  did 
not  obtain  constant  and  uniform  results,  be- 
cause as  we  now  know  they  did  not  recognize 
the  necessity  of  regulating  the  size  and  fre- 


PROTEIK    FEVER  77 

quency  of  the  doses.  In  1909  my  students  and 
I  showed  that  by  regulating  the  amount  and 
frequency  of  the  dosage  we  could  induce  any 
desired  form  of  fever,  acute,  fatal,  intermit- 
tent, remittent  or  continued. 

Inasmuch  as  I  have  given  elsewhere*  the  de- 
tails of  this  work  I  will  only  reproduce  the  con- 
clusions and  make  a  few  general  remarks:  (1) 
Large  doses  of  unbroken  protein  administered 
intraabdominally,  subcutaneously  or  intrave- 
nously have  no  effect  on  temperature,  at  least 
do  not  cause  fever.  (2)  Small  doses,  especially 
when  repeated,  cause  fever,  the  forms  of  which 
may  be  varied  at  will  by  changing  the  size  and 
frequency  of  the  dosage.  (3)  The  effect  of  pro- 
tein injections  on  the  temperature  is  more 
prompt  and  marked  in  sensitized  than  in  fresh 
animals.  (4)  The  intravenous  injection  of 
laked  blood  corpuscles  from  either  man  or  the 
rabbit  causes  in  the  latter,  even  in  small  quan- 
tity, either  in  single  or  repeated  doses,  prompt 
and  marked  elevation  of  temperature.  (5) 
Laked  corpuscles  after  removal  of  the  stroma 
by  nitration  have  a  like  effect.  (6)  Protein  fe- 
ver can  be  continued  for  weeks  by  repeated  in- 
jections, giving  a  curve  which  cannot  be  dis- 
tinguished from  that  of  typhoid  fever.    (7)  Pro- 

*Protein  Split  Products  in  Relation  to  Immunity  and  Disease,  Lea  & 
Febiger,  1913. 


78  POISONOUS   PROTEINS 

tein  fever  is  accompanied  by  increased  nitrogen 
elimination  and  gradual  wasting,  (8)  Protein 
fever  includes  most  instances  of  clinical  fever. 
(9)  Animals  killed  by  experimentally  induced 
fever  may  die  at  the  height  of  the  fever,  but 
as  a  rule  the  temperature  falls  rapidly  before 
death.  (10)  Fever  induced  by  repeated  injec- 
tions of  bacterial  proteins  and  ending  in  re- 
covery may  be  followed  by  immunity.  (11)  The 
serum  of  animals  in  which  protein  fever  has 
been  induced  digests  the  homologous  protein  in 
vitro.  In  view  of  recent  work  on  antif erment 
in  blood  serum  this  point  needs  reinvestigation. 
(12)  Fever  is  one  of  the  results  of  the  parenteral 
digestion  of  proteins.  (13)  There  are  two  kinds 
of  parenteral  proteolytic  enzymes,  one  specific 
and  the  other  nonspecific.  (14)  The  production 
or  activation  of  the  nonspecific  ferment  is  easily 
and  quickly  stimulated.  (15)  The  development 
of  the  specific  ferment  requires  a  longer  time. 
(16)  Sensitization  and  lytic  immunity  are  dif- 
ferent manifestations  of  the  same  process.  (17) 
Foreign  proteins,  living  or  dead,  formed  or  in 
solution,  when  introduced  into  the  blood  soon 
diffuse  through  the  tissues  and  sensitize  the 
cells.  Different  proteins  have  predilection 
places  in  which  they  are  deposited  and  where 
they  are,  in  large  part  at  least,  digested,  thus 


PROTEIN   FEVER  79 

giving  rise  to  the  characteristic  symptoms  and 
lesions  of  the  different  diseases.  (18)  The  sub- 
normal temperature  which  may  occur  in  the 
course  of  a  fever  or  at  its  termination  is  due  to 
the  rapid  liberation  of  the  protein  poison  which 
in  small  doses  causes  an  elevation,  and  in 
larger  doses  a  depression  of  temperature.  (19) 
Fever  per  se  must  be  regarded  as  a  beneficent 
phenomenon  inasmuch  as  it  results  from  a 
process  inaugurated  by  the  body  cells  for  the 
purpose  of  ridding  the  body  of  foreign  sub- 
stances. (20)  The  evident  sources  of  excessive 
heat  production  in  fever  are  the  f ollowing :  (a) 
that  arising  from  the  unusual  activity  of  the 
cells  supplying  the  enzyme;  (b)  that  arising 
from  the  cleavage  of  the  foreign  protein;  (c) 
that  arising  from  the  destructive  reaction  be- 
tween the  split  products  from  the  foreign  pro- 
tein and  the  proteins  of  the  body. 

The  above  are  the  conclusions  which  I  drew 
three  years  ago  from  experiments  which  my 
students  and  I  had  carried  out  and  from  a 
study  of  the  literature  of  the  subject.  I  did  not 
suppose  at  the  time,  nor  do  I  hold  now,  that  all 
these  conclusions  are  exactly  right. 

The  fundamental  fact  that  the  parenteral  in- 
troduction of  proteins  may  induce  fever  is 
founded  upon  so  many  independent  observa- 


80  POISONOUS   PKOTEINS 

tions,  some  of  them  recorded  many  years  ago, 
that  I  do  not  think  it  incumbent  upon  me  to 
seek  additional  support.  Friedberger  has,  in 
a  most  exact  way,  confirmed  the  statement  that 
large  doses  of  foreign  protein  do  not,  while 
small  doses  do  elevate  the  temperature.  More- 
over, he  has  shown  that  a  small  dose  is  more 
effective  in  sensitized  than  in  unsensitized  ani- 
mals. 

Thiele  and  Embleton  have  confirmed  experi- 
mentally the  proposition  that  the  parenteral  in- 
troduction of  foreign  proteins  affects  the  tem- 
perature, causing  a  rise  or  fall  or  having  no 
effect  according  to  the  size  of  the  dose.  They 
give  the  following  tables: 

Egg-White. 

normal  animal        sensitized 

Limits  of  grams  grams 

Temperature    fall 0.05  0.005 

Constant    temperature 0.02  0.0002  to  0.0001 

Temperature    rise 0.01  to  0.001  0.0001  to  0.000002 

Tubercle  Emulsion. 

normal  animal        sensitized 
Limits  of  grams  grams 

Temperature    fall 0.005  to  0.002         0.0005 

Constant  temperature. 0.002  to  0.001         0.0001 
Temperature    rise 0.001  to  0.00001     0.00001  to  0.000001 

Criticism  of  the  statement  that  foreign  pro- 
teins find  certain  predilection  tissues  in  which 
they  accumulate  has  been  made.     Iodine  ac- 


PROTEIN   FEVER  81 

cumulates  in  the  thyroid  gland.  Mercury  in- 
duces characteristic  lesions  in  the  kidneys. 
Strychnia  selects  a  definite  portion  of  the  nerv- 
ous tissue  on  which  its  action  is  made  manifest. 
The  therapeutic  effects  of  the  most  approved 
drugs  depend  upon  their  predilection  for  cer- 
tain tissues.  The  recent  studies  of  Eosenow 
indicate  that  bacterial  proteins  do  not  differ 
from  other  poisons  in  this  respect.  We  are  ac- 
customed to  think  of  chemotaxis  as  acting  only 
between  morphologically  recognizable  bodies, 
but  in  reality  it  is  a  form  of  chemism  and  is  de- 
pendent upon  chemical  composition  and  not  on 
histological  structure. 

The  only  one  of  the  above  given  statements 
formulated  some  years  ago  which  has  met  with 
any  experimental  negation  is  my  contention 
that  specific  proteases  are  developed  by  the  pa- 
renteral introduction  of  foreign  proteins.  I  am 
ready  to  admit  that  Friedberger's  anaphyla- 
toxin  comes  from  the  serum.  In  fact  at  the 
same  time  that  I  formulated  the  proposition 
concerning  protein  fever  I  wrote  as  follows: 
"It  has  been  suggested:  (a)  That  the  agar  or 
kaolin  or  bacteria  absorb  the  complement  from 
the  serum  and  that  this  renders  it  poisonous, 
(b)  That  the  poison  is  preformed  in  the  serum, 
but  that  its  action  is  neutralized  by  some  other 


82  POISONOUS   PROTEINS 

constituent  of  the  serum  which  is  absorbed  by 
the  agar  or  kaolin,  (c)  That  the  absorption  of 
some  constituent  of  the  serum  by  the  agar,  kao- 
lin or  bacteria  leads  to  a  disturbance  of  the 
equilibrium  of  the  protein  constituents  of  the 
serum  which  as  a  consequence  break  up  with 
the  liberation  of  the  poison.  These  suggestions 
assume  that  the  poison  comes  from  the  serum 
and  this  may  be  true. ' '  On  another  page  I  said : 
"That  the  anaphylatoxin  comes  from  the  blood 
serum,  the  one  constant  factor  in  all  the  experi- 
ments in  its  production,  is  most  probable.' ' 
Now  since  the  probability  has  become  a  cer- 
tainty, we  need  not  conclude  that  specific  pro- 
teases never  result  from  the  parenteral  intro- 
duction of  proteins.  I  have  shown  that  all  pro- 
teins, including  those  of  blood  serum,  contain  a 
poison  and  I  am  not  at  all  surprised  on  learning 
that  such  a  poison  in  the  serum  is  set  free  in  the 
production  of  Friedberger's  anaphylatoxin  and 
in  the  development  of  Abderhalden's  preg- 
nancy test,  but  these  have  nothing  to  do  with 
the  development  of  proteases  in  smallpox  or  ty- 
phoid fever.  At  least  no  such  connection  has 
been  shown. 

The  Phenomena  of  Infection. 

I  have  elsewhere  gone  into  some  detail  con- 
cerning the  views  of  the  nature  of  infection 


THE   PHENOMENA   OF   INFECTION  83 

which  I  have  developed  in  my  studies  on  the 
chemistry  and  toxicology  of  bacterial  and  other 
proteins.  Only  a  living  thing  can  infect.  In- 
jection of  diphtheria  or  tetanus  toxin  may 
cause  all  the  symptoms  and  lesions  of  the  re- 
spective diseases,  but  such  injections  are  arti- 
ficial procedures  and  the  results  are  intoxica- 
tions rather  than  infections.  In  this  section  I 
shall  omit  diseases  due  to  toxins.  The  infect- 
ing agent  is  a  virus  and  in  infections  there  is  a 
contest  between  the  invader  and  the  native.  It 
is  a  struggle  for  food,  growth,  and  reproduc- 
tion. In  the  bacterial  diseases  the  structure  or 
the  equipment  of  the  invader  is  quite  as  compli- 
cated and  as  complete  as  that  of  the  defender. 
The  contest  is  between  bacterial  and  body  cells 
and  the  battlefield  may  involve  only  a  small 
part  or  may  extend  to  every  part  of  the  ani- 
mal's body. 

What  is  the  difference  between  pathogenic 
and  nonpathogenic  bacteria?  In  order  for  a 
given  bacterium  to  be  pathogenic  to  a  given 
animal  it  must  be  possible  for  the  former  to 
feed  upon  the  latter.  All  living  things  feed  by 
means  of  digestive  ferments.  Continued  life  and 
multiplication  are  impossible  under  other  con- 
ditions. First,  in  order  for  a  given  bacterium 
to  infect  a  given  animal  the  ferments  of  the 


84  POISONOUS   PROTEINS 

former  must  be  able  to  digest  the  proteins  of 
the  animal.  In  the  second  place  the  invading 
cells  must  not  be  immediately  destroyed  by  the 
ferments  elaborated  by  the  body  cells.  There 
must  be  a  supporting  relation  between  the  bac- 
terial cell  and  the  medium,  and  in  infection  the 
body  constitutes  the  medium  in  which  the  bac- 
teria grow  and  multiply.  The  protein  groups 
split  from  the  medium  must  fit  into  the  molecu- 
lar structure  of  the  bacterial  cell;  otherwise 
they  would  be  of  no  service  to  it.  Many  kinds 
of  cells  may  live  in  the  same  medium,  but  for 
each  kind  the  cleavage,  of  the  medium  must  be 
specific.  From  this  it  follows  that  the  agent  by 
which  the  cleavage  products  are  secured  must 
be  supplied  by  the  cell  and  must  be  specific  to  it. 
It  follows  from  what  has  been  said  that  a 
bacterium  placed  in  a  medium  in  which  its  fer- 
ment is  ineffective  cannot  grow  and  multiply. 
A  bacterium  which  cannot  grow  and  multiply 
in  the  animal  body  cannot  cause  an  infection. 
Its  inability  to  grow  and  multiply  in  the  animal 
body  may  be  due  to  the  fact  that  its  ferments 
cannot  digest  or  properly  break  up  the  proteins 
of  the  animal  body.  This  is  one  of  the  reasons 
why  the  great  majority  of  bacteria  are  harm- 
less or  nonpathogenic.  This,  however,  is  not 
the  sole,  and  probably  not  the  dominant  cause 


THE   PHENOMENA   OF   INFECTION  85 

of  the  failure  of  so  many  species  of  bacteria  to 
do  harm  to  the  higher  animals.  What  has  been 
said  about  the  production  and  utilization  of 
ferments  by  the  bacterial  cell  is  equally  true 
of  the  body  cell.  In  fact,  it  is  true  of  every  liv- 
ing cell.  The  body  cell  has  its  specific  ferments, 
and  the  bacterial  cell  being  protein  substance  is 
liable  to  be  digested  by  the  ferments  elaborated 
by  the  body  cells.  In  these  simple  facts  lies  the 
fundamental  explanation  of  all  forms  of  bac- 
terial immunity,  either  natural  or  acquired.  It 
will  be  understood  that  I  am  here  omitting  all 
reference  to  the  elaboration  of  toxins  and  anti- 
toxins. 

Ferments  are  intra-  and  extracellular.  All 
are  formed  within  the  cell,  but  some  diffuse  into 
the  medium  while  others  do  not.  In  some  in- 
stances at  least,  cell  permeation  by  the  pabulum 
is  essential  to  the  feeding  of  the  cell.  In  other 
cases  the  ferment  accumulates  on  the  surface 
where  digestion  proceeds.  In  others  the  fer- 
ment diffuses  into  the  medium  more  or  less 
widely  from  the  cell  which  produces  it.  Many 
cells  produce  both  intra-  and  extracellular  fer- 
ments, and  these  differ  in  function. 

I  am  not  going  into  detail  concerning  cellular 
ferments.  Those  of  the  bacterial  cells  are  easi- 
ly obtained  and  have  been  studied  quite  elabo- 


86  POISONOUS   PKOTEINS 

rately.  Some  digest  proteins,  such  as  gelatine, 
quickly  while  others  are  less  prompt  and  others 
still  have  no  recognizable  effect  on  this  protein. 
They  are  easily  affected  by  the  presence  of 
certain  nonprotein  substances,  especially  car- 
bohydrates. The  ferments  of  the  body  cells  are 
not  so  easily  obtained  and  are  more  difficult  of 
study.  However,  both  the  intra-  and  extra- 
cellular ferments  of  the  polymorphonuclear 
corpuscles  have  been  studied  in  some  detail  and 
their  destructive  action  on  certain  bacteria  has 
been  demonstrated.  The  germicidal  action  of 
the  blood  and  its  serum  has  been  demonstrated 
on  various  species  of  bacteria. 

It  may  be  well  to  point  out  some  differences 
between  intra-  and  extracellular  ferments.  The 
latter  are  comparable  to  the  enzymes  of  the  ali- 
mentary canal.  Their  function  is  solely  a  lytic 
one.  They  break  up  complex  proteins  into  sim- 
pler bodies,  but  these  without  further  treat- 
ment are  not  ready  to  be  built  into  the  cellular 
structure.  The  extracellular  ferments  are  in  a 
general  way  destructive  in  action.  The  intra- 
cellular ferments  are  essentially  constructive. 
They  shape  the  rough  blocks  and  fit  them  into 
the  molecular  structure.  In  the  process  of  in- 
fection the  intracellular  ferments  of  the  bacte- 
rial cells  are  most  active.    The  soluble,  simple 


THE   PHENOMENA   OF   INFECTION  87 

proteins  of  the  fluids  of  the  animal's  body  are 
quickly  built  into  the  bacterial  cell  and  growth 
and  multiplication  result.  Body  proteins  are 
converted  into  bacterial  proteins.  This  process 
proceeds  so  smoothly  that  as  a  rule  during  the 
time  when  its  development  is  most  rapid  the 
host  is  quite  unaware  of  the  presence  of  his  un- 
desired  guest.  Whole  molecules  of  albumins 
and  globulins  are  taken  into  the  bacteria  and 
built  into  the  more  complicated  bacterial  cell. 
This  is  the  period  of  incubation  in  an  infection. 
The  body  cells  are  not  prepared  to  combat  the 
invader  during  this  period.  Finally  the  body 
cells  react  and  begin  the  elaboration  of  fer- 
ments which  destroy  the  bacterial  proteins. 
This  is  quite  a  different  process.  Complex, 
cellular  proteins  are  split  into  simpler  ones  and 
protein  poisons  are  set  free. 

During  the  period  of  incubation  of  an  infec- 
tious disease,  the  infecting  organism  supplies 
the  ferment,  the  simple,  soluble  proteins  of  the 
body  fluids  constitute  the  substrate,  the  process 
is  essentially  constructive,  no  poison  is  set  free 
and  there  are  no  recognizable  clinical  symp- 
toms. During  the  active  progress  of  an  infec- 
tious disease,  the  body  cells  supply  the  ferment, 
the  complex,  bacterial,  cellular  proteins  consti- 
tute the  substrate,  the  process  is  essentially  de- 


88  POISONOUS   PROTEINS 

structive,  the  protein  poison  is  set  free,  the 
symptoms  of  disease  appear,  lesions  more  or 
less  destructive  develop  and  life  is  placed  in 
jeopardy. 

The  experienced  clinician  will  easily  "under- 
stand that  in  most  infections  diseases  the  steps 
in  the  evolution  of  the  processes  are  not  so 
clearly  denned  as  indicated  in  the  above  state- 
ments.   They  are  most  typical  in  uncomplicated 
cases  of  yellow  fever,  typhoid  and  typhus  and 
in  smallpox,  but  even  in  these  there  often  are 
complicating  factors.     In  yellow  fever  an  at- 
tempt is  made  to  eliminate  the  poison  into  the 
alimentary  canal  as  is  evidenced  by  black  vomit. 
In  typhoid  the  poison  in  being  excreted  into  the 
intestine  may  lead  to  perforation.    In  most  in- 
fections, the  bacterial  growth  and  their  disrup- 
tion overlap.    In  one  part  of  the  body  the  bac- 
teria continue  to  grow  while  in  other  parts  they 
are  being  destroyed.    In  pneumonia  life  may 
be  endangered  by  the  abundance  and  extent  of 
the  exudate,  while  in  the  crisis  of  this  disease 
autolysis  probably  plays  an  important  role  not 
only  in  the  destruction  of  the  organisms,  but  in 
the  removal  of  the  exudate.    In  many  infections 
lesions  develop  and  impair  the  efficiency  of  the 
body  cells.    Moreover  in  destructive  lesions  the 
dead  tissues  of  the  body  must  be  disposed  of 


THE   PHENOMENA   OF   INFECTION  89 

and  this  throws  an  increased  burden  on  the 
body  cells.  In  some  diseases  phagocytosis 
plays  an  important  role.  It  must  be  evident 
that  the  engnlfment  of  bacteria  by  phagocytes 
is  a  more  conservative  method  of  disposing  of 
the  invading  cells  than  their  extracellular  de- 
struction, since  in  the  former  the  body  is  pro- 
tected against  the  poison  liberated  by  bacterial 
cleavage.  Nothing  more  dangerous  to  the  in- 
fected individual  could  happen  than  the  sud- 
den cleavage  of  all  the  bacteria  in  his  body. 
The  poison  liberated  in  this  process  would 
overwhelm  him  at  once.  This  is  a  probable  ex- 
planation of  the  fact,  already  referred  to,  that 
the  case  mortality  in  typhus  fever  is  higher 
among  the  well  nourished  than  among  the  less 
robust.  Bacterial  cells,  as  well  as  body  cells, 
have  means  of  protecting  themselves.  The  tu- 
bercle bacillus  through  limitless  generations  of 
parasitism  has  developed  coatings  of  fats  and 
waxes  which  protect  it  against  the  action  of 
secretions  of  body  cells  quite  as  efficiently  as 
coats  of  mail  protected  our  ancestors  against 
the  weapons  of  their  time.  Moreover,  bacterial 
cells  may  develop  increased  resistance  or  be- 
come to  some  extent  immune  to  the  action  of 
body  cell  secretions.  Occasionally  bacteria  per- 
sist in  the  body  for  long  periods  after  recovery 


90  POISONOUS   PROTEINS 

from  the  disease  and  when  these  are  trans- 
ferred to  new  hosts  they  show  that  they  have 
lost  nothing  in  virulence.  Frequently,  second- 
ary infections  develop  and  decide  the  fate  of 
the  individual.  As  someone  has  said  the  pyo- 
genic microorganisms  frequently  play  the  last 
act  in  the  great  tragedies  of  life,  tuberculosis, 
cancer,  and  syphilis. 

A  Chemico-Biologic  Concept  of  the  Protein 
Molecule. 

Under  this  heading  I  wish  to  formulate  cer- 
tain theories  which  have  developed  in  my  mind 
during  the  progress  of  the  work  which  I  have 
outlined  in  preceding  lectures.  Some  men 
seem  able  to  work  without  developing  theories 
and  probably  this  is  best,  but  I  have  never 
worked  in  that  way.  It  is  possibly  a  fault;  if 
it  be,  I  am  ready  to  confess  that  I  have  sinned 
and  continue  in  the  same  old  way.  I  hope  that 
some  of  the  statements  which  I  am  about  to 
make  will  stimulate  others  to  investigate  and 
this  I  deem  of  more  importance  than  their  truth 
or  falsity. 

The  protein  poison  about  which  all  my  work 
has  centered  is  a  fact.  It  has  been  prepared 
and  studied  by  so  many  competent  men  that  its 
wide    distribution    in   proteins    from    diverse 


THE   PROTEIN"    MOLECULE  91 

sources  cannot  be  questioned.  Its  effects  on 
animals  have  been  widely  tested  and  the  gen- 
eral conclusions  reached  are  quite  as  uniform 
as  those  which  might  be  formulated  about  poi- 
sons much  longer  known.  Its  chemical  struc- 
ture has  not  been  determined  with  certainty. 
The  best  evidence  at  hand  today  seems  to  indi- 
cate that  it  is  not  a  basic  body,  and  therefore 
not  a  protein  alkaloid,  not  a  leucomain  or  a 
ptomain.  It  contains  no  phosphorus  and  no 
carbohydrate.  In  the  purest  form  in  which  it 
has  been  obtained,  it  yields  a  trace  of  ash  of 
which  phosphorus  and  chlorine  are  not  essen- 
tial constituents.  Whether  this  mineral  matter 
is  an  essential  part  of  the  poison  or  not,  I  do 
not  know.  Under  any  condition  in  which  it  has 
been  obtained  it  is  decidedly  acid  in  character 
and  yields  amino  acids  on  disruption.  It  seems 
to  be  a  polypeptid. 

Underhill,  whose  opinion  I  esteem  highly, 
concludes  that  the  action  of  the  protein  poison 
on  animals  is  similar  in  kind  but  more  intense 
than  that  of  proteoses.  I  dare  say  that  this  is 
quite  right  and  it  conforms  with  my  own  ob- 
servations. I  suggested  in  the  Shattuck  lec- 
tures in  1906  that  the  protein  poison  is  the 
chemical  nucleus,  keystone,  or  archon  of  larger 
and  more  complicated  protein  molecules. 


92  POISONOUS   PROTEINS 

The  chemism  of  the  protein  poison  is  intense 
and  it  combines  with  various  inorganic  and  or- 
ganic sub  stances  to  form  more  complex  mole- 
cules, still  retaining  and  imparting  to  these 
larger  molecules  its  protein  characteristics. 
Combined  with  phosphate  of  lime  it  forms  such 
phosphoglobulins,  so-called,  as  casein.  Com- 
bined with  carbohydrate  it  develops  the  glyco- 
proteins and  in  combination  with  both  phos- 
phorus and  carbohydrates,  the  glyco-nucleo- 
proteins  result.  In  the  last  mentioned  bodies 
the  protein  molecule  reaches  its  most  complex 
form,  and  further  development  is  possible  only 
by  polymerization  and  the  aggregation  of  many 
protein  molecules  into  cells.  At  what  stage  in 
the  evolution  of  the  protein  molecule  metabo- 
lism begins  I  cannot  say,  but  it  is  quite  evident 
that  multiplication  does  not  begin  until  the 
most  complex  structure  has  been  reached.  It 
seems  quite  evident  that  from  the  beginning  the 
process  is  a  synthetical  one. 

It  is  possible  to  conceive  of  the  beginning  of 
life  on  the  earth,  as  proceeding  in  this  way.  In 
the  intense  heat  of  past  geological  ages  when 
even  carbon  existed  in  the  gaseous  state  this 
element  combined  with  nitrogen  forming  cyano- 
gen. With  this  binary  compound  under  proper 
conditions  the  synthesis  of  the  simplest  amino 


THE   PROTEIN   MOLECULE  93 

acid  was  possible  for  cyanogen  may  react  with 
boiling  hydroiodic  acid  with  the  development  of 
amino  acetic  acid  and  from  this  the  other  amino 
acids  f onnd  in  the  protein  molecule  might  have 
been  developed.  In  this  view,  proteins  in  their 
simplest  form  may  have  come  into  existence 
long  before  life  as  we  now  know  it  was  pos- 
sible on  the  earth. 

The  simplest  protein,  as  the  protein  poison, 
has  its  intense  chemism  satisfied  as  it  combines 
with  other  elemental  gronps  in  the  development 
of  the  more  complex  bodies. 

I  began  my  work  with  the  hope  of  finding 
simple  proteins  in  the  cellular  structures  of 
bacteria.  In  this  I  was  disappointed  and  I  now 
see  that  I  shonld  not  have  expected  it.  Instead 
of  finding  simple  proteins  in  bacterial  cells  I 
have  fonnd  them  in  the  casein  of  milk  and  in 
the  proteoses  of  seeds.  As  I  have  already  said 
the  young  mammalian  is  fed  npon  food  prin- 
ciples served  in  the  simplest  form.  The  nurs- 
ing child  is  supplied  with  fats  as  such,  with 
mineral  constituents  for  the  most  part  uncom- 
bined,  with  carbohydrates  in  the  easily  assimi- 
lable form  of  lactose  and  with  amino  acids  in  the 
relatively  simple  protein,  casein.  The  sprouting 
seed  finds  the  amino  acids  with  which  it  starts 
life  in  the  relatively  simple  proteins  while  fats 


94  POISONOUS  PROTEINS 

and  carbohydrates  are  supplied  in  a  ready- 
made  form.  Now  if  this  provision  be  made  for 
the  support  of  the  developing  plant  and  animal, 
what  can  be  said  about  the  food  supplied  the 
numerous  cells  of  the  body,  whether  it  be  plant 
or  animal.  Simple  proteins  exist  in  the  circu- 
lating blood  of  the  higher  animals.  Not  only 
is  this  true  but  as  Van  Slyke  and  his  co-workers 
have  shown  the  body  cells  directly  use  amino 
acids.  The  simple  proteins  probably  exist  in 
the  circulating  blood  chiefly  in  that  protein  mix- 
ture about  which  we  know  but  little  and  which 
we  designate  as  serum  globulin.  In  this  mix- 
ture the  primitive  proteins  are  ready  to  enter 
into  combination  with  the  more  complex  cellu- 
lar proteins  as  the  latter  wear  away  in  their 
functional  activities.  Their  chemism  is  held  in 
abeyance  by  combination  with  some  indifferent 
substances,  such  as  calcium.  I  have  found  that 
the  protein  poison  from  casein  is  neutralized  in 
vitro  by  calcium  lactate.  Indeed  the  protein 
poison  is  largely,  but  not  so  quickly,  neutralized 
by  incubation  with  sodium  bicarbonate.  In  this 
connection  it  may  be  well  to  recall  the  effect  of 
the  withdrawal  of  calcium  on  the  coagulation 
of  blood  and  that  after  severe  poisoning  with 
the  protein  body  the  clotting  of  the  blood  is  re- 
tarded and  often  wholly  prevented.    If  my  idea 


THE   PKOTELN"    MOLECULE  95 

that  the  circulating  Mood  at  all  times  contains 
the  protein  poison  from  the  too  violent  chemism 
of  which  the  body  is  normally  protected  by  its 
combination  with  an  inert  body  is  correct,  it  will 
not  be  difficult  to  understand  that  the  equilib- 
rium may  be  disturbed  in  a  variety  of  ways  with 
death  as  a  result.'  The  introduction  of  a  little 
more  of  the  poison  or  the  removal  of  the  protect- 
ing body  may  seriously  upset  the  equilibrium. 
Casein  yields  about  ninety  per  cent  of  its 
weight  in  protein  poison.  The  calcium  is  easily 
removed  from  casein.  An  ash-free  casein  may 
be  prepared  by  repeated  solution  in  dilute  am- 
monia and  reprecipitation  with  dilute  acid.  The 
last  trace  of  calcium  is  removed  by  treatment 
with  oxalic  acid.  The  protein  poison  from 
casein  resembles  the  globulins  inasmuch  as  it 
may  be  wholly  precipitated  from  aqueous  solu- 
tion by  saturation  with  sodium  chloride,  but  dif- 
fers from  globulins  inasmuch  as  it  is  freely  sol- 
uble in  absolute  alcohol. 

Blood  is  rendered  poisonous  not  only  by  in- 
cubation with  bacteria,  agar,  starch,  kaolin,  etc., 
but  as  was  shown  by  Kohler  as  long  ago  as 
1877,  it  becomes  poisonous  on  clotting,  killing 
both  homologous  and  heterologous  animals. 
This  phenomenon,  which  has  been  confirmed  by 
others,  has  recently  been  investigated  by  Mol- 


96  POISONOUS   PROTEINS 

dovan  who  has  shown  that  blood  freshly  defi- 
brinated  by  shaking  with  glass  beads  causes 
acute  death  when  injected  intravenously  into 
guinea-pigs  and  rabbits.  In  the  former  the 
typical  anaphylactic  lung  picture  is  seen  after 
death.  When  the  dose  is  slightly  sublethal 
there  is  marked  fall  in  temperature  with  subse- 
quent fever.  When  the  doses  are  smaller  there 
is  marked  fever.  On  standing  from  fifteen  to 
forty-five  minutes  defibrinated  blood  loses  its 
toxicity.  Serum  obtained  by  rapid  centrifuga- 
tion  of  defibrinated  blood  is  poisonous.  The 
same  is  true  of  the  deposited  and  once  washed 
corpuscles.  When  coagulation  is  delayed  by 
the  presence  of  sodium  citrate  neither  the  su- 
pernatant fluid  nor  the  corpuscles  are  poison- 
ous, but  both  become  so  when  coagulation  is  in- 
duced by  shaking  with  porcelain  beads.  Do  err 
has  shown  that  blood  received  in  paraffined  ves- 
sels becomes  poisonous ;  but  when  the  coagula- 
tion is  complete  the  toxicity  disappears.  When 
coagulation  is  made  to  proceed  slowly  by  the 
addition  of  hirudin  solution  or  a  0.7  per  cent 
solution  of  colloidal  silicic  acid,  it  retains  its 
toxicity  for  several  hours. 

The  fact  that  extracts  of  normal  tissue,  when 
injected  intravenously,  are  poisonous  is  another 
interesting  fact.    If  the  lungs  of  a  rabbit  be 


THE   PKOTEIX    MOLECULE  97 

macerated  for  two  hours  in  salt  solution,  the 
solution  kills  promptly  on  intravenous  injec- 
tion. Homologous  organ  extracts  are  more 
poisonous  than  heterologous. 

All  these  phenomena  show  that  there  is  un- 
der normal  conditions  a  nice  adjustment  in  the 
constituents  of  blood  and  tissue  whereby  life  is 
protected  and  that  slight  changes  easily  disturb 
this  equilibrium  with  most  disastrous  results. 
There  are  here  unsolved  problems  but  my  work 
leads  me  to  the  conclusions  that  there  are  pro- 
tein bodies  in  the  blood  and  tissue,  which  serve 
under  normal  conditions  as  cell  foods,  but 
which  may  become  explosively  poisonous  when 
the  mechanism  regulating  their  use  is  dis- 
turbed. Normal  cells  contain  deposits  of  these 
bodies,  which  under  proper  regulation,  supply 
cell  waste,  but  under  abnormal  conditions  lead 
to  cell  destruction.  These  substances  were 
probably  present  in  my  bacterial  cells,  but  I 
washed  them  out  and  threw  them  away  leaving 
only  the  cellular  proteins.  However,  time  and 
labor  will  solve  these  problems  and  I  turn  to 
another  phase  of  my  subject. 

If  I  properly  interpret  my  work  on  the 
chemistry  of  bacterial  proteins  it  confirms  the 
theoretical  views  of  Pfhiger,  Ehrlich,  and  Ver- 
worn,  who  have  held  that  the  essential  part  of 


98  POISONOUS   PROTEINS 

cells  consists  of  a  chemical  unity,  made  up  of 
giant  molecules.  So  far  as  I  can  find,  this  view 
receives  additional  support  in  the  experimental 
work  done  by  others.  I  have  been  able  to  find 
but  little  upon  this  subject.  Eeinke  and  Bode- 
wald  found  that  air  dried  substance  of  sethy- 
lium  septicum,  which  they  designate  as  Plasmo- 
dium, consists  largely  of  highly  complex  pro- 
teins containing  phosphorus  and  yielding  xan- 
thine bases  and  carbohydrates  on  disruption. 
Sosnowski  concludes  from  his  study  of  infuso- 
rial cellular  substance  that  this  does  not  con- 
tain simple  proteins  as  such,  but  as  constitu- 
ents of  highly  complex  molecules.  My  studies 
have  led  me  to  formulate  a  theory  concerning 
the  nature  and  operation  of  living  matter.  My 
first  attempt  in  this  direction  was  made  in  a 
lecture  delivered  in  Toronto  (1905),  and  this 
was  elaborated  in  a  Shattuck  lecture  (1906). 
The  cell  is  not  the  unit  of  life ;  life  is  molecular. 
Life  is  function,  not  form.  The  cell  is  not  only 
made  up  of  protein  molecules,  but  its  form  and 
function  are  determined  by  the  chemical  struc- 
ture of  its  constituent  molecules.  The  lines 
along  which  the  spore,  seed  or  ovum  develop 
are  determined  by  the  chemical  structure  of  its 
proteins.  Growth  in  other  directions  is  impos- 
sible, and  this  accounts  for  stability  in  repro- 


THE   PROTEIN    MOLECULE  99 

duction.  However,  changes  in  the  chemical 
structure  may  and  do  occur  and  in  these  lies  the 
basis  of  variation. 

The  keystone  or  archon  of  the  protein  mole- 
cule is  the  protein  poison.  It  is  common  to  all 
protein  molecules.  Physiologically  it  is  the 
same  in  all  molecules ;  i.  e.,  when  set  free  it  is  a 
poison  and  it  is  a  poison  on  account  of  its  in- 
tense chemism  which  enables  it  to  tear  ofT 
groups  from  other  proteins.  One  protein  dif- 
fers from  another  in  its  secondary  and  tertiary 
groups.  Most  native  proteins  are  not  poison- 
ous because  in  them  the  chemism  of  the  pri- 
mary group  is  satisfied  by  combination  with 
secondary  groups.  Strip  off  the  secondary 
groups  and  the  primary  becomes  poisonous  on 
account  of  the  avidity  with  which  they  combine 
with  the  secondary  groups  of  other  molecules. 
Biological  relationship  between  proteins  de- 
pends upon  the  secondary  groups.  In  this  way 
varieties  and  species  have  developed. 

The  living  molecule  is  never  in  a  state  of 
equilibrium.  There  is  a  constant  exchange  of 
atoms  between  it  and  the  outside  world.  It  ab- 
sorbs, assimilates  and  eliminates.  It  is  con- 
stantly trading  in  energy.  It  takes  in  oxygen 
and  gives  off  carbonic  acid;  it  takes  in  nitrog- 
enous material,  and,  having  utilized  it,  the  waste 


100  POISONOUS   PROTEINS 

is  discarded.  The  living  molecule  passes 
through  the  period  of  growth  and  decay.  Dur- 
ing the  former,  its  functions  are  largely  syn- 
thetic ;  in  the  latter  they  are  autolytic  and  final- 
ly the  structure  drops  into  pieces. 


PAET  IV. 

THE  PUBITY  OF  ALCOHOL. 

In  my  work  with  the  protein  poison,  I  have 
found  it  necessary  to  give  attention  to  the 
purity  of  the  alcohol  used  and  I  have  found  the 
alcohol  imported  from  Germany  (Kahlbaum's) 
not  always  up  to  the  standard.  In  fact,  I  have 
found  it  cheaper  and  safer  to  take  the  ordinary 
alcohol  and  distill  it  with  quick  lime.  The  fol- 
lowing tests  may  be  used  in  determining  the 
purity  of  alcohol. 

1.  The  specific  gravity  is  determined  by  the 
picknometer. 

2.  It  should  be  miscible  in  all  proportions 
without  cloudiness  with  water,  ether  and  chlo- 
roform. 

3.  It  should  not  redden  litmus  even  after  four 
hours'  exposure.  Kahlbaum's  alcohol  often 
reddens  litmus  in  a  much  shorter  time. 

4.  The  evaporation  of  50  c.c.  should  leave  no 
weighable  residue.  Some  samples  of  Kahl- 
baum's alcohol  gave  in  this  amount  as  much 
as  1  mg.  and  some  more. 

5.  A  mixture  of  10  c.c.  of  the  alcohol,  5  c.c. 


102  POISONOUS   PROTEINS 

of  water  and  1  c.c.  of  glycerine,  allowed  to 
evaporate  spontaneously  on  clean  blotting  pa- 
per, leaves  no  foreign  odor  when  the  last  trace 
of  the  alcohol  has  disappeared.  This  is  a  U.  S. 
P.  test  and  shows  the  absence  of  more  than  a 
trace  of  fusel  oil. 

6.  A  mixture  of  10  c.c.  of  the  alcohol  and  0.2 
c.c.  of  two  per  cent  KOH  solution  is  evapo- 
rated to  1  c.c.  and  then  treated  with  an  excess 
of  dilute  (1:4)  sulphuric  acid.  This  should  not 
develop  the  odor  of  fusel  oil. 

7.  10  c.c.  of  the  alcohol  is  evaporated  to  2 
c.c.  and  this  is  shaken  with  an  equal  volume  of 
sulphuric  acid.  The  development  of  a  reddish 
color  shows  the  presence  of  amylic  alcohol. 

8.  When  20  c.c.  of  the  alcohol  is  shaken  in  a 
clean  glass  stoppered  bottle  with  1  c.c.  of  silver 
nitrate,  test  solution,  the  mixture  should  not 
become  more  than  faintly  opalescent  or  acquire 
more  than  a  faint  brownish  tint  when  exposed 
to  diffuse  daylight  for  six  hours.  This  is  a 
U.  S.  P.  test  for  organic  impurities,  amylic  al- 
cohol, aldehyde,  etc.  The  imported  alcohols  are 
not  up  to  standard  at  all  times  by  this  test. 

9.  Into  a  test  tube  which  has  been  rinsed  with 
the  alcohol,  pour  5  c.c.  of  sulphuric  acid,  then 
layer  the  acid  with  an  equal  amount  of  the  alco- 
hol.   The  appearance  of  a  red  zone  after  stand- 


PURITY   OF   ALCOHOL  103 

ing  for  four  hours  or  longer  indicates  the  pres- 
ence of  molasses  alcohol.  Neither  domestic  nor 
foreign  alcohols  respond  to  this  test. 

10.  Pass  hydrogen  sulphide  gas  through  20 
c.c.  of  the  alcohol  for  from  two  to  five  minutes, 
then  add  a  few  drops  of  ammonia  and  allow  to 
stand  for  four  hours.  The  alcohol  should  re- 
main colorless.  If  it  becomes  yellowish  or 
brownish,  the  presence  of  traces  of  metal,  ex- 
tractives or  tannin  is  indicated.  By  this  test 
many  of  the  samples  of  imported  alcohol  are 
shown  not  to  be  up  to  standard. 

11.  To  10  c.c.  of  the  alcohol  add  1  c.c.  of  a 
solution  of  potassium  permanganate  (1-1000). 
Allow  to  stand  twenty  minutes,  the  develop- 
ment of  a  yellowish  or  brownish  color  indicates 
the  presence  of  aldehyde.  Many  of  the  im- 
ported alcohols  show  this  impurity  in  more 
than  traces.  Our  redistilled  alcohol  does  not 
show  it. 

12.  To  5  c.c.  of  the  alcohol  add  two  drops  of 
a  one  per  cent  aqueous  solution  of  furfurol,  un- 
derlay this,  kept  cold  in  a  stream  of  water,  with 
5  c.c.  of  sulphuric  acid.  The  formation  of  a 
colored  zone,  gradually  becoming  pink,  shows 
the  presence  of  amylic  alcohol.  This  is  an  ex- 
ceedingly delicate  test  and  by  making  a  stand- 
ard solution  of  amylic  alcohol  the  proportion 


104  POISONOUS   PEOTEINS 

can  be  approximated.  Kahlbaum's  alcohol,  as 
we  have  found,  often  shows  appreciable  traces 
of  amylic  alcohol  by  this  test. 

13.  To  10  c.c.  of  the  alcohol  add  ten  drops  of 
colorless  analin,  then  three  drops  of  hydro- 
chloric acid.  No  coloration  shonld  develop 
within  five  niinntes.  This  is  a  test  for  furfurol, 
which  we  have  not  f  onnd  in  either  domestic  or 
imported  alcohols. 

14.  Evaporate  100  c.c.  of  the  alcohol  to  dry 
ness,  extract  the  residue  with  from  3  c.c.  to  5 
c.c.  of  salt  solution  and  inject  this  intravenous- 
ly into  a  guinea-pig  of  from  200  g.  to  300  g. 
weight.  With  our  redistilled  alcohol  there  is  no 
effect,  while  with  Kahlbaum's  the  animal  is 
often  thrown  into  convulsions  which  may  ter- 
minate fatally.  What  this  residue  contains,  I 
have  not  been  able  to  determine.  It  is  granular 
and  sometimes  contains  a  few  needle  shaped 
crystals. 

From  my  experience,  I  have  found  it  better 
to  use  alcohol  prepared  in  my  own  laboratory 
from  the  commercial  ninety-five  per  cent 
article,  by  redistillation  with  caustic  lime,  than 
to  depend  upon  the  imported  product. 


PAET  V. 

TISSUE   CELLULAR  PROTEIN  POISONS. 

Cunmiings  and  Chambers*  have  prepared  the 
protein  poison  from  varions  organs  and  tis- 
sues and  have  tested  its  effects  npon  both  homo- 
logons  and  heterologous  animals.  They  de- 
scribe their  methods  of  preparing  the  tissue  as 
follows :  Tissues  including  muscle,  brain,  heart, 
lungs,  liver,  pancreas,  and  kidney  were  obtained 
from  the  dog,  goat,  ox,  and  rabbit.  Because 
the  protein  poison  can  be  obtained  from  blood 
constituents  it  was  necessary  to  remove  these 
before  clotting  occurred,  thus  securing  organs 
free  from  water  soluble  protein.  The  follow- 
ing procedure  was  carried  out  to  gain  this  re- 
sult with  the  dog,  goat,  and  rabbit :  The  ani- 
mal was  first  anesthetized,  then  a  glass  cannula 
was  introduced  into  the  abdominal  aorta;  the 
jugulars  were  then  cut  and  water  was  forced 
under  mild  pressure  into  the  circulatory  sys- 
tem. Several  liters  of  wash  water  were  used 
in  each  case,  the  washing  being  continued  until 


*Cummings  and   Chambers:     Jour.   Lab.   and   Clin.   Med.,  i,  428. 


106  POISONOUS   PROTEINS 

tlie  fluid  running  from  the  jugulars  had  lost  its 
red  tinge.  The  ox  organs  were  removed  im- 
mediately after  death,  and  each  was  washed 
free  from  blood  by  forcing  water  through  its 
circulatory  system.  The  organs  were  cut  into 
half-inch  cubes  and  washed  in  water  with  fre- 
quent changes ;  the  final  wash  water  gave  none 
of  the  tests  for  protein.  The  tissues  were  then 
dehydrated  in  80  per  cent  and  100  per  cent 
alcohol  each  for  twenty-four  hours.  They  were 
then  dried  and  powdered  with  a  fine  meat 
grinder. 

The  tissue  thus  prepared  was  split  into  poi- 
sonous and  nonpoisonous  parts  by  a  2  per  cent 
solution  of  sodium  hydroxide  in  absolute  alco- 
hol. The  poisonous  products  were  tested  out 
on  animals.    Conclusions  are  stated  as  follows : 

1.  Vaughan's  protein  poison  can  be  prepared 
from  tissue  cells  of  the  exsanguinated  organs 
of  multicellular  animals. 

2.  The  tissue  cellular  protein  poisons  are  not 
only  toxic  for  heterologous  species,  but  also  for 
homologous  species. 

3.  The  M.L.D.  of  the  protein  poisons — here 
reported — for  the  guinea-pig  and  the  rabbit  is 
in  proportion  to  their  relative  body  weights 
when  given  by  the  intraperitoneal  method  of  in- 
jection; when  given  intravenously,  however,  it 


COLOR   REACTIONS   AND   SPLIT   PRODUCTS      107 

is,  in  proportion  to  body  weight,  twenty-five 
(25)  times  more  toxic  for  the  guinea-pig  than 
the  rabbit. 

4.  Tissue  cellular  protein  poison  hastens  the 
clotting  of  the  blood  of  the  guinea-pig,  rabbit, 
and  dog  in  vivo.  The  protein  poison  prepared 
from  casein  differs  from  these  in  that  it  either 
retards  or  prevents  entirely  the  clotting  of  dog's 
blood. 

5.  Witte's  peptone  does  not  prevent  the  clot- 
ting of  rabbit's  blood  in  vitro. 

6.  The  in  vitro  experiments  here  reported 
show  that  all  the  protein  poisons  tested  inhibit 
the  clotting  of  blood  from  the  guinea-pig,  rab- 
bit and  dog,  in  certain  percentages. 

THE  COLOR  EEACTIONS  OF  PROTEINS 
AND  THEIR  SPLIT  PRODUCTS. 

Emerson  and  Chambers*  have  made  a  com- 
parative study  of  the  protein  color  reactions 
on  the  original  proteins  and  their  poisonous 
and  nonpoisonous  split  products.  These  stud- 
ies give  some  conception  of  the  lines  along 
which  the  cleavage  of  the  protein  develops. 
The  conclusions  are  stated  as  follows: 

1.  The  proteins  and  their  split  products,  the 


'Emerson  and   Chambers:      Jour.    Lab.    and    Clin.    Med.,    i,   692. 


108  POISONOUS   PROTEINS 

protein  poisons  and  the  residues,  all  give  the 
binret  reaction.  The  residues  will  not  give  the 
reaction  in  dilutions  much  greater  than  1-100. 
This  indicates  that  the  proteins  and  their  split 
products  contain  an  acid  amide  group  and  other 
substituted  amide  groups  attached  to  neighbor- 
ing carbon  atoms,  and  that  the  end  products 
have  not  been  deamidized  in  cleaving. 

2.  Gies'  biuret  reagent  gives  similar  results. 

3.  The  proteins  and  their  split  products  all 
give  the  xanthoproteic  reaction,  and  the  poi- 
sons, in  greater  dilutions  than  the  proteins  or 
the  residues,  indicating  that  they  all  contain 
benzene  nuclei  and  that  in  cleaving,  these  tend 
to  concentrate  in  the  poison. 

4.  The  residues  do  not  give  Millon's  reaction 
and  the  poisons  give  it  in  greater  dilutions  than 
the  proteins,  indicating  that  all  the  mono-hy- 
droxy-benzene  nuclei  (tyrosine)  are  cleaved  off 
into  the  poison.  It  is  interesting  to  note  the 
strong  reaction  given  by  the  colon  cell  substance 
and  by  the  poison. 

5.  The  residues  do  not  give  Bardach's  reac- 
tion and  all  soluble  proteins  are  said  to  give  it. 
The  residues  are  alkaline  and  soluble. 

6.  The  poisons  do  not  give  the  Molisch  reac- 
tion and  the  residues  give  a  stronger  Molisch 
than  the  proteins,  indicating  that  the  carbohy- 


COLOR   REACTIONS   AND    SPLIT   PEODUCTS      109 

drate  groups  are  cleaved  off  into  the  residues. 
Casein  gives  a  very  weak  Molisch  reaction,  and 
casein  residue  a  strong  reaction;  this  may  be 
due  to  tryptophane,  but  we  think  not,  as  the 
casein  residue  gives  only  a  weak  tryptophane 
reaction.  Casein  residue  does  not  reduce  Fehl- 
ing's  solution  even  after  boiling  with  dilute  hy- 
drochloric acid.  Part  of  the  residue  is  insolu- 
ble in  10  per  cent  alkali  and  this  insoluble  part 
gives  the  Molisch  reaction  and  also  tests  for 
nitrogen. 

7.  The  proteins  and  their  split  products  all 
give  the  Aclamkiewicz  reaction  indicating  that 
they  all  contain  the  tryptophane  group.  Solu- 
tions of  tyrosine  give  pinkish  violet  colors  which 
are  similar  to  the  tryptophane  reactions  in  di- 
lute solutions. 

8.  The  protein  poisons  do  not  give  good  Hop- 
kins-Cole reactions  when  performed  in  the  reg- 
ular way.  The  proteins  and  residues  do.  They 
all  give  good  positive  reactions  when  furfural 
is  added.  The  protein  poisons  probably  give 
negative  reactions  because  they  contain  no  car- 
bohydrate. 

9.  Benedict's  modification  of  Hopkins-Cole 
reagent  gives  similar  results  both  with  and 
without  furfural. 

10.  The  Acree  Eosenheim  formaldehyde  test 


110  POISONOUS   PKOTEINS 

gives  similar  results  to  those  obtained  with  the 
Hopkins-Cole  reagent,  both  with  and  without 
furfural. 

11.  The  proteins  and  the  residues  give  posi- 
tive results  with  the  benzaldehyde  test  (Reichl's 
reaction).  The  poisons  give  negative  reactions 
and  the  addition  of  furfural  has  no  effect. 

THE  NINHYDRIN  REACTIONS. 

The  same  investigators  have  made  a  study  of 
the  ninhydrin  test  as  applied  to  the  original 
protein  and  the  split  products.  This  reaction 
has  become  prominent  on  account  of  its  delicacy 
and  because  of  its  use  in  the  Abderhalden  test. 

The  conclusions  in  this  study  show  that  the 
poisonous  split  product  gives  this  reaction  in 
the  same  dilution  as  the  original  protein,  while 
the  nonpoisonous  part  fails  to  respond  even  in 
highly  concentrated  solution.  Emerson  and 
Chambers  state  their  results  as  follows: 

1.  Vaughan's  protein  poisons  in  dilutions  up 
to  1-10,000  give  the  ninhydrin  reaction. 

2.  The  proteins  from  which  the  poisons  are 
obtained  will  also  give  the  ninhydrin  reactions 
in  dilutions  up  to  1-10,000. 

3.  The  cell  residue  in  dilutions  of  1-100  does 
not  give  the  ninhydrin  reaction. 


PROTEIN  skin  reaction  111 

4.  Dilute  acids  and  alkalis  interfere  with  this 
reaction. 

5.  Sodium  chloride  interferes  very  slightly 
with  the  reaction. 

6.  Prolonging  the  time  of  heating  makes  the 
reaction  very  much  more  delicate. 

THE  PEOTEIN  SKIN  REACTION. 

Much  use  has  been  made  in  recent  years  of 
certain  skin  reactions  for  diagnostic  purposes. 
Such  is  the  von  Pirquet  test  in  tuberculosis.  I 
have  long  held  that  this  is  a  protein  reaction. 
The  tubercular  individual  elaborates  in  his  body 
cells  a  ferment  which  splits  up  tuberculin  and 
sets  the  protein  poison  free,  and  the  reaction  is 
due  to  the  local  effects  of  the  liberated  protein 
poison  on  the  epithelial  cells.  This  explanation 
is  confirmed  by  recent  observations.  A  man 
claimed  that  he  could  not  eat  even  a  small 
amount  of  any  food  containing  nutmeg  without 
soon  becoming  sick  and  developing  urticarial 
rash.  A  bit  of  nutmeg  was  crushed  in  a  mortar 
in  a  few  drops  of  sterile  salt  solution  and  a  drop 
of  this  suspension  placed  on  the  forearm  and 
scratched  in  with  a  von  Pirquet  scarafier.  With- 
in a  few  minutes  a  large  wheal  or  hive  developed 
about  the  point.  Similar  tests  have  been  made 
with  similar  results  on  those  sensitive  to  pep- 


112  POISONOUS   PROTEINS 

tone,  egg  white,  etc.  The  explanation  is  fur- 
ther confirmed  by  the  demonstration  that  the 
protein  poison  liberated  by  chemical  agents  in 
vitro  produces  a  like  effect  upon  all  cells.  By 
this  simple  procedure  the  sensitiveness  of  per- 
sons to  certain  proteins  may  be  demonstrated 
and  the  degree  roughly  approximated.  Pos- 
sibly sensitiveness  to  horse  serum  might  be 
tested  in  this  way  preliminary  to  the  adminis- 
tration of  diphtheria  antitoxin,  but  I  have  not 
known  of  its  being  tried. 


INDEX 

Alcohol,  purity  of,  101 

Kahlbaum's,  not  always  satisfactory,  101 

tests  for  determining  purity  of,  101 
Animal  proteins,  47 
Autolytic  cleavage: 

effects  on  foreign  proteins,  55 

of  bacterial  proteins,  56 

of  cholera  bacillus,  57 

of  proteins,  53 

of  unicellular  proteins,  56 
Autolytic  enzymes,  54 
Bacterial  cellular  protein,  13 

action  on  animals,  37 

animal  experimentation  with,  30 

chemistry  of,  14 

poisonous  action  of,  30 

preparation  of  material,  13 

split  products  of,  34 
Bacterial  cellular  substances: 

carbohydrates,  presence  of,  16 

cellulose,  presence  of,  15 

chitin,  presence  of,  16 

molecules  in,  19 

nuclein,  presence  of,  17 

proteins  detached  by  alkalis  and  acids,  IS 
Blood,  how  rendered  poisonous,  95 
Cellular  proteins,  action  on  animals,  41 
Cellulose  in  bacteria,  15 

Chemico-biologic  concept  of  the  protein  molecule,  90 
Chemistry  of  bacterial  cellular  protein,  30 
Cleavage  of  proteins,  53 
Color  reactions  and  their  split  products,  107 
Crude  soluble  poison,  35 

action  on  animals,  42 


114  INDEX 

Ferments,  intra-  and  extra-cellular,  85 
Infection,  phenomena  of,  82 
Kahlbaum's  alcohol,  101 

Living  colon  bacillus,  action  on  animals,  37 
Ninhydrin  reactions  of  proteins  and  their  split  products,  110 
Parenteral  introduction  of  foreign  products,  effect  on  tem- 
perature, 80 
Parenteral  protein  digestion,  58 

Pathogenic  and  nonpathogenic  bacteria,  differentiated,  83 
Peptone  immunity,  49 
Peptone  poisoning,  49 
Phenomena  of  infection,  82 

Poisonous  action  of  bacterial  cellular  protein,  13 
Protein  digestion,  parenteral,  58 

parenteral  and  enteral,  differentiated,  58 
Protein  fever,  75 

Protein  molecule,  chemico-biologic  concept  of,  90 
Protein  poison,  35 

action  on  animals,  37 

tests  for,  36,  91 

tissue  cellular,  105 
Protein  skin  reactions,  111 
Proteins  and  split  products: 

color  reactions  of,  107 

ninhydrin  reactions  of,  110 

skin  reactions  of,  111 
Proteins: 

animal,  47 

autolytic  cleavage  of,  53 

bacterial  cellular,  13 

vegetable,  45 
Proteoses,  48 
Specific  proteoses  in  sensitized  animals,  summary  of  work 

on,  71 
Split  products  of  bacterial  cellular  proteins,  34 
Tamura's  contribution  to  the  chemistry  of  bacteria,  21 
Tissue  cellular  protein  poisons,  105 
Vegetable  proteins,  45 


Valuable     Medical    Books 

The  Newer  Methods  of 
Blood  and  Urine  Chemistry 

By  E.  B.  H.  GRADWOHL,  M.D.,  Director  of  The  Pasteur 
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Laboratory  Methods 


By  B.  G.  R.  WILLIAMS,  M.D.,  and  E.  G.  C.  WILLIAMS, 
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Valuable     Medical    Books 

Journal  of  Laboratory  and 
Clinical  Medicine 

VICTOR  C.  VAUGHAN,  M.D.,  Editor-in-Chief,  Ann  Arbor, 
Mich.  Associate  Editors :  D.  E.  JACKSON,  M.D.,  St.  Louis; 
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Experimental  Pharmacology 

By  DENNIS  E.  JACKSON,  Ph.D.,  M.D.,  Associate  Professor 

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C.  V.  Mosby  Company — Publishers — St.  Louis 


COLUMBIA  UNIVERSITY  LIBRARIES 

This  book  is  due  on  the  date  indicated  below,  or  at  the 
expiration  of  a  definite  period  after  the  date  of  borrowing, 
as  provided  by  the  rules  of  the  Library  or  by  special  ar- 
rangement with  the  Librarian  in  charge. 

DATE  BORROWED 

DATE  DUE 

DATE  BORROWED 

DATE  DUE 

1 

IAY  2  9  1943 

.urn 

1-fi  iq*7 

AUG  '< 

II  1948 

NO 

f  K        :  *-: 

fkj 

v  \) 

C28M  I  AO)  M  100 

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